Ramy Salameh reports on how Rotterdam is harnessing its port city zest to reinvent its events proposition.
Rotterdam is home to the world's first floating farm, with 40 cows, a farmer and robots soon to be collecting manure and feeding cattle; while the city's innovation district garners and aligns start-ups, incubators and accelerators with institutions and businesses.
All of this activity swirls around Europe's largest (and smartest) port, which is working towards becoming the first C02 neutral port in the world, part of the authorities' 'energy transition' strategy set for 2030.
Rotterdam sees itself as a Living Lab and a focal point of urban innovation and sustainability. It is a city that encourages experimental projects spanning everything from the 'circular economy' to 'Resilience' and is one of the Rockefeller Foundation's 100 Resilient Cities.
Rotterdam's youthful vibe and zest is also evident in the cutting-edge architectural landmarks that dominate the skyline.
The city's arts and culture scene saw it host the 20th edition of 'Art Rotterdam Week' recently, part of a year-round programme of festivals and events.
It is no surprise, in this context, that The Netherland's second city has been capturing some big MICE wins and turning heads away from the capital, Amsterdam.
Meeting planners are clearly seeing something new, raw and relevant when considering the city. "Rotterdam being a 'smart city' is an incentive, but as The Netherlands prides itself on green initiatives and other cities such as Amsterdam and Copenhagen are also well known for their sustainable practices, this in itself is not enough.
The differentiator with Rotterdam is its drive for sustainability, combined with its unique architecture and urban vibe, and lower costs compared to more popular destinations," says Paul Stoddart, Managing Director, CWT Meetings & Events, UK, Ireland & Benelux.
What has limited Rotterdam before, in terms of capacity to host the mega-events, is now being addressed with new venue developments.
Rotterdam Ahoy will add a new Congress and Convention centre in late 2020 and will feature The Netherlands' largest auditorium, which, when combined with the existing halls and arena, has already launched Rotterdam into a wider bidding pool for conventions and tradeshows.
A recent win to come off the back of Ahoy's development is the 'ASME Turbo Expo Conference 2022', focusing on turbines and energy.
The 1930's Van Nelle Factory, the primary venue for Art Rotterdam Week, received its UNESCO World Heritage status in 2014, a rarity for a former factory complex.
This vision can be seen in the way art is appreciated in Rotterdam, with the arrival of 'Bojimans Van Beuningen Depot' in early 2021 in the Museumpark. It will be the world's first publicly accessible art storage facility, housing Museum Bojimans Van Beuningen's collection of 151,000 artworks.
In 2016, Rotterdam's modernist cultural appeal saw Lonely Planet vote it a 'Best in Travel' top-10 city and, shortly after, accessibility to the city, was boosted by the arrival of Eurostar into Centraal Station. The city was also chosen to be the European seat of Boston's Cambridge Innovation Centre and the home of the UN Global Centre of Excellence for Climate Adaptation.
The climate for conferencing in Rotterdam is certainly looking rather more clement.


PTC Batteries have access to an extremely large range of forklift traction batteries and accessories.
As this is an extremely specialised field we have found the easiest and most convenient way to give you a quote is for you to contact us and we can call to your site to measure up your old batteries etc. and send the information to our traction battery department.
They will then reply with a price within a couple of days.


Every employee wants to impress his/her bosses to stay in their good books. And there's nothing wrong with that! It constantly pushes you to deliver your best work and proactively work towards achieving organizational goals. You may want to help the boss whenever asked for, but you also need to draw a line somewhere. In your attempt to become the go-to person for the boss, you will often find yourself feeling overworked and stressed. In the long run, this will start affecting your productivity, and your overall reputation at the office will also suffer.
If you've been feeling overworked lately, sit down and analyze the daily tasks you're involved in. Chances are the amount of time you spend on extra tasks exceeds the time you spend on the tasks you were initially hired for. This is a sign that it's time to start saying no to taking on extra work. Saying no to the boss, especially when you have never said so earlier, takes courage. However, it's not impossible if done the right way.
This article lists five polite, yet effective ways to say no to your boss without offending him/her or sabotaging your work reputation.
Your boss probably keeps giving you more work because they don't know about all the other tasks you have at hand. This is especially true if you have multiple people assigning work to you. In that case, it's impossible for the boss to be aware of all the work you already have on your plate. Had s/he known earlier s/he wouldn't have bothered you in the first place.
The next time your boss asks you to work on an additional project, just inform him/her about your current schedule. Instead of bluntly saying no, just say that you'll get back to him/her on the new project as soon as you can as you are currently occupied with other projects and have deadlines to meet.
Whether you do an additional task or not is secondary. What's primary is to express your appreciation to the boss for considering you capable of the task.
It's always better to start a conversation on a positive note. Show that you are enthusiastic about the new work/project and would love to assist after you complete your current job tasks.
Is it just your boss who assigns tasks and projects to you? Great! Despite giving you a number of tasks with set deadlines, s/he asks you to take on additional work. Chances are either s/he has forgotten about the tasks already assigned to you or the priority has changed.
It's important to communicate openly with your boss and discuss the priority of each task assigned to you. Once you're clear on this, you'll be able to work more productively without feeling burdened or worried about missing deadlines.
You may have too much on your plate already and simply don't have the bandwidth to give immediate attention to a new task. However, stating your own problems (even when they are genuine) to the boss when s/he is seeking help from you is definitely not the right approach.
Bosses appreciate problem-solvers, and even if you can't assist directly, you can help in other ways. For example, you can propose to share your input instead of completing the whole task yourself or suggest a colleague's name whom you know is eager to take on additional work responsibilities.
As humans, we love being acknowledged and heard. The same applies to your boss too. Appreciate the opportunity and explain to him/her your current work deadlines. Discuss the new work requirements with your boss and buy some time to respond to the request. Understanding what they want you to do will help you gauge how much time you'll have to dedicate to the new project. You'll establish an effective dialogue with your boss regarding your daily work commitments and how they'll be affected after taking up the additional work.
Setting healthy boundaries in the workplace is important to avoid unnecessary stress and excessive workload. Saying no to your boss won't ruin your reputation as a hard worker if you do it the right way. Instead, it will establish your reputation as a high-value employee who is good at prioritizing tasks and believes in delivering quality work within deadlines.


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Home»2017»01 - January»NWRI graduate Fellows announced
01 - January
NWRI graduate Fellows announced
The National Water Research Institute has named five Graduate Fellows who have been awarded $10,000 (USD) to support their research projects related to developing and enhancing water supplies. Natalie Hull is a second-year doctoral student at University of Colorado-Boulder, working on Mechanisms and Sustainability of Wavelength-Tailored Ultraviolet Drinking Water Disinfection for Small Systems. Mojtaba Azadi Aghdam is a first-year doctoral student pursuing a degree in environmental engineering at the University of Arizona. His research, A Novel Brine Precipitation with the Aim of Higher Water Recovery, aims to increase the clean water recovery rate from membranes using brine precipitation. Second-year doctoral student (University of Wisconsin-Madison) Devon Manley's research project, Study of the Viability of Chlorine Photolysis as an Advanced Oxidation Process in Water Treatment Systems, aims to determine the optimum treatment conditions (i.e., pH, irradiation wavelength and chlorine concentration) for contaminant removal by chlorine photolysis. Fifth-year doctoral student (University of Colorado-Boulder) Kyle Shimabuku's dissertation, Biochar Sorbents for the Control of Organic Contaminants: Understanding Biochar Structure and Water Quality on Sorption Behavior, focuses on developing sustainable and low-cost adsorbents for stormwater and wastewater treatment and reuse. Jenna Krichling is pursuing her Master's Degree in environmental health at San Diego State University. Her research project, Non-Targeted Analysis for Discovery of Chemicals of Emerging Concerns in Treated Water for Drinking and Source Investigation, will help ensure wastewater introduced into bodies of water is safe to later be reclaimed and treated for human consumption. The NWRI Fellowship program is underwritten by the Joan Irvine Smith & Athalie R. Clarke Foundation, community partners and NWRI Corporate Associates.
Calibration rig service
Vertical, multi-stage inline pumps

redpajama_set_name: RedPajamaCommonCrawl


On Day 4 of the second Test in Bengaluru Australia needed to score 188 runs to take a 2-0 lead in the four-match Test series. The guests had made an exciting rebound on the fourth day of the Bengaluru Test, taking six wickets for 61 with the second new ball. The Australians without a doubt had a high ground in the Test match yet India demonstrated praiseworthy character and came up with the best when it mattered most. There were a couple of things that India did right and here are a look at five of them.
Cheteshwar Pujara and Ajinkya Rahane shared an unbeaten 93-run partnership to take India to 213/4 at stumps on Day 3. This partnership was most needed for India as it moved them to a lead of 187 for Australia.
Both put forth a concentrated effort splendidly in the center and played with a great determination. This was just the third 50 run partnership for India in the two Tests.
The pace assault of Umesh Yadav and Ishant Sharma played their heart out. Their spells kept India in the match. Ishant, who is an amazing talent, got in the faces of the batsmen and demonstrated a considerable measure of rage while Yadav swung the ball at energetic pace. It was Umesh Yadav who got the crucial breakthrough when he packed away the wicket of Steve Smith in the second innings. Together the pacers scalped 5 crucial Australian wickets and that too on a pitch which favoured the spinners.
Virat Kohli gave Ravindra Jadeja just 21.4 overs in the Australian innings however the spinner completed with 6 wickets for 63 runs. This was a match winning spell. R Ashwin bowled 49 overs in the first innings. Umesh Yadav and Ishant Sharma bowled 24 and 27 overs individually and the three bowlers shared four wickets among them.
However, Ravindra Jadeja bowled the minimal overs in the Australian innings yet he got six wickets for 63 runs and that too in only 21.4 overs. Jadeja's six wickets includeded that of three left handers. For the most part, a left-arm spinner won't be more effective against left-arm batsmen. Be here is Jadeja, who demonstrated why he is India's top bowler. It was Jadeja's bowling that helped India to limit Australia to 276 in their first innings and their lead was just 87 runs.
Indian opener, KL Rahul demonstrated splendid intent when he came out to bat in both the innings, especially 35 minutes before lunch in the second innings. He checked his common impulses to score 90 from 205 balls, acknowledged that the wicket at the M. Chinnaswamy Stadium was a bit challanging, and had some dampness right off the bat. Overall it was a great knock which shows his ability to perform in pressure, with great determination.
Steve Smith record of scoring against India has been incredible and as a general rule he has been a thistle in India's mission for victory. So if India wanted to win the Test match against Australia then they have to figure out how to dismiss captain Steven Smith early. More often Smith has ended up being India's adversary, pacing his innings to flawlessness. However, in this match he could only manage the scores of 8 and 28 in the both the innings.


Donahoe: I think there's no doubt digital currency is going to play an important role going forward, and at paypal, we're going to have to integrate digital currencies into our wallet.
Lipton: So handicap it. How quickly do you think we'll see bitcoin in Paypal?
Donahoe: I don't know, I can't give a specific time. But I think you'll see Bitcoin used in different use cases, whether it's a peer-to-peer use case, a cross-border transaction, someone's sending currency to someone else, and over time, you'll start to see it with some merchants accepting Bitcoin. And I don't know how big it will get, how quickly, but the underlying theme is a digital coin something we should be aware of.
Lipton: Do you own any bitcoin, personally?
Lipton: What do you think it should be worth?
Donahoe: For me, I'm not buying it as an investment. I'm buying it to understand how it can be used. I think there are two sides of it, the investment side and the digital currency side. I'm more interested in the digital currency side- about how you and I can exchange value seamlessly using technology.
Lipton: I don't know if it's a Stockholm syndrome or what, but everybody that I talk to out here, and you sort of get into it, really does talk about Bitcoin as the next big thing. And they believe it. And they mean it. It is not just some kind of fly-by-night thing they are playing with. They are serious and very big companies out here, very, very serious about it.
In acknowledging that he owns Bitcoin, he joins colleague David Marcus, CEO of PayPal, who indicated the same when reassuring the public about their stance on Bitcoin in January.
Bitcoin has just spiked by over 3% in less than an hour, now trading near $660 on Bitstamp. One can certainly speculate that the jump came in response to Donahoe's comments, which came to light within the past hour. While the causes for Bitcoin's rise over the past few weeks remain unclear, it may indeed be a result of positive sentiment of tech giants finally getting into digital currency.


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A Pawfect Stud (filed in Rated G, Short Snorts on May 8, 2019)
Hope Urine the Mood for a Potty Pun (filed in Rated PG, Short Snorts on May 22, 2018)
A Mess of Food Puns (filed in Gatling Puns, Rated G on January 25, 2018)
Punniest of Show 2017: Haley Haradan (filed in Gatling Puns, Video on May 18, 2017)
Punniest of Show 2017: Arum Debray (filed in Gatling Puns, Video on May 17, 2017)
Sister Cities (filed in Puns, Rated PG on May 9, 2016)
Windows of Opportunity (filed in Rated G, Shaggy Dogs on September 9, 2015)
Just a Couple of Dandylions (filed in Picture Pun, Rated G, Short Snorts on August 25, 2015)
Sex Antics (filed in Rated G, Short Snorts on February 12, 2015)
Just Duckserts (filed in Rated PG, Shaggy Dogs on December 22, 2014)
If he'd been missing a fifth appendage, they would have gone to a retailer. (filed in Rated G on October 1, 2014)
A Future CAWnvict (filed in Picture Pun, Rated G on May 8, 2014)
So, Zoo Me! (filed in Gatling Puns, Picture Pun, Rated G on April 8, 2014)
Not a Boaring Advertisement (filed in Picture Pun, Rated PG on April 7, 2014)
A Pun for the Halibut (filed in Puns, Rated PG-13, Short Snorts on January 14, 2014)
I Dream of Jeannie She's a Light Brown Hare (filed in Rated G on January 3, 2014)
Dublin my Fortune (filed in Rated G, Shaggy Dogs on November 19, 2013)
A Knight to Remember (filed in Rated G, Shaggy Dogs on November 18, 2013)
Ducktor Who? (filed in Rated G, Shaggy Dogs on October 15, 2013)
Fossilzzle (filed in Picture Pun, Puns, Rated G on July 25, 2013)
There is a Frog in his Throat (filed in Rated G, Short Snorts on May 23, 2013)
The Cow, the Ant, and the Old Fart (filed in Rated PG-13, Short Snorts on April 22, 2013)
Hoof in Mouth Disease (filed in Rated PG, Short Snorts on February 6, 2013)
Cuddle Up, and Don't Be Blue (filed in Picture Pun, Rated G on January 24, 2013)
Wisdom for the New Year (filed in Picture Pun, Rated G on January 8, 2013)


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Imelda Anne Crawford (22 November 1920 – 17 October 1956) was a British film actress, born in Palestine of a Scottish father and an English mother, and brought up in Edinburgh.

Biography
A contemporary of Margaret Lockwood and Phyllis Calvert, Crawford is best remembered for her roles in women's pictures of the 1940s, such as Millions Like Us (1943), Two Thousand Women (1944) and They Were Sisters (1945).

She married Wallace Douglas in 1953 and died of leukaemia in London in 1956, aged 35. The Times, on 18 October 1956, reported that she was playing in Agatha Christie's The Spider's Web, at London's Savoy Theatre, when she became ill. Also, that after acting in a stage production of The Gift, about a scientist blinded by an accident, she added a codicil to her will leaving her eyes to the International Eye Bank.

Filmography

Film

Television

References

External links

1920 births
1956 deaths
Scottish actresses
British film actresses
Deaths from leukemia
Deaths from cancer in England
20th-century British actresses

redpajama_set_name: RedPajamaWikipedia


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The St Jerome in Penance is a painting by the Italian Renaissance painter Filippo Lippi, dating to c. 1439. It is housed in the Lindenau-Museum of Altenburg, Germany. The work could be identified with the St. Jerome Penitent of which Lippi asked payment in a letter issued to Piero de' Medici in 1439.

The painting is one of the first known example on the subject of St. Jerome doing penance. It is a small panel, divided into two different scenes: in the upper part is St. Jerome with the wooden cross, the stone to strike his breast and the lectern lying on the rock. In the lower part, with a chronological step, is the episode of the saint with the lion having a thorn in the paw. The unfriendly landscape is a metaphor of the hermit style of life.

1430s paintings
Paintings by Filippo Lippi
Lippo
Lions in art
Skulls in art


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Q: Find statement in matlab and multiple conditions The following code is embedded in a function
rsp = find(response_times >= current1 & response_times < current2 & response_times ~= current2);

Here, I am looking for the indices of responses that occur between current1 and current2, where current1 and current2 are times such as 16.22 and 16.32, respectively, and the response times can be equal to current1 but not current2.
For the most part this works as intended, however, every so often it pulls an index of a value equal to current2.
Does anyone know why this might be the case or how I can improve this one line of code to fix it.
Here is an example array this code operates on:
response_times = [ 8.73000000000000
    11.4300000000000    13.4800000000000
    14.7900000000000    16.3200000000000
    18.0400000000000    20.3800000000000
    20.9900000000000    21.3400000000000
    24.2800000000000    24.6800000000000 ];

A: It's not actually (exactly) equal to current2.  You generally shouldn't compare floating point numbers for equality. Read this article for more information, but the essence of the problem is that most values cannot be represented exactly with a floating point representation (i.e.  IEEE 754), hence it is not advisable to test for equality. Inequalities are fine. There is actually a neat mini-site dedicated to providing a basic explanation of the issue.
For the Stack Overflow matlab version of the explanation see this Q&A, entitled "Why is 24.0000 not equal to 24.0000 in MATLAB?". It's quite an interesting read!
To verify that your inequality is actually working, have it compute abs(response_times-current2) and you should find that the value is not zero in those cases, but rather something small like 1.35e-15. If you want to reject these "too close" values, include a test such as (current2-response_times)>tol and set tol to something large enough to reject these points.

redpajama_set_name: RedPajamaStackExchange


text:
[clappr](../../../index.md) / [io.clappr.player.plugin.control](../../index.md) / [TimeIndicatorPlugin](../index.md) / [Companion](index.md) / [entry](./entry.md)

# entry

`val entry: `[`Core`](../../../io.clappr.player.plugin/-plugin-entry/-core/index.md)

redpajama_set_name: RedPajamaGithub


The first option is the easiest: take it to an AT&T Wireless store (or Verizon/Sprint Store for CDMA iPhones), set up a new account, and receive a new SIM card. A second option is to jailbreak the iPhone and use it as an iPod touch without phone service. A third option is to jailbreak how to pack for travel abroad 2. Unfold the paper clip and push one end into the hole until the tray slides out. Alternatively, use the SIM eject tool if you received one with your phone.
10/08/2014 · Solved can I activate a new sim card into my old phone I need a new phone number Forum Solved Want a new android smartphone Forum I need to activate my new sim card with.a new number.after store my pc keeps opening new tabs how to stop this 2. Unfold the paper clip and push one end into the hole until the tray slides out. Alternatively, use the SIM eject tool if you received one with your phone.


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China's RMB on the world stage, now & tomorrow
Michelle Zhu 30/09/2016, 10:35am
SINGAPORE (Sept 30): Starting Saturday Oct 1, China's currency will be included in the International Monetary Fund's (IMF) official foreign exchange reserves database.
This comes nearly a year after the organisation gave the nod on Nov 30, 2015, for the renminbi (RMB) to join the US dollar, the euro, the pound sterling and the Japanese yen in its Special Drawing Rights (SDR) basket.
(See also: Yuan IMF reserves entry seen as more symbolic than game-changing)
Guy Harvey-Samuel, CEO of HSBC Singapore, declares the move as a "harbinger of the Chinese yuan's elevation onto the world stage", and a "clear recognition of its development as a global medium of exchange".
Rising role among central banks
"Whilst SDR inclusion does not automatically translate into reserve-currency status, its role within central banks is becoming more pronounced," says Harvey-Samuel.
He highlights how HSBC's latest annual Reserve Management Trend survey reflects a 10-fold increase in central banks that are now investing in the RMB since 2012 to 32. The study also projects that the currency's share of global reserves is expected to rise to 7% in 2020 and to 10% in 2025.
"The RMB's inclusion has also reignited market interest in SDR-denominated instruments: the World Bank recently issued an SDR bond in China that was settled in CNY," says the CEO. "In the long run, this will help to enhance the role of the SDR in the international monetary system."
What the inclusion means for Singapore
Harvey-Samuel believes the resulting boost in confidence and long-term demand for the RMB is likely to further improve the currency's liquidity and stability in Singapore.
(See also: MAS to count renminbi investments as part of official foreign reserves)
"Already a RMB stronghold, Singaporean businesses' ability to tap into a deeper pool of liquidity will have a positive multiplier in their ability to capture future inbound Chinese investment, including those linked to China's Belt and Road initiative," he asserts.
Next step for the RMB
Meanwhile, Paul Mackel, HSBC Global Research's head of emerging markets (EM) foreign exchange (forex) research, says the onshore-offshore basis in the USD-RMB's forwards are in focus again.
In a Thursday report, he notes that there has been high volatility in the China Offshore Spot (CNH) forex forwards/swaps market, with "sharp divergence" between the onshore-offshore curves although the onshore USD-China Renminbi (CNY) spot exchange rate has been "more or less stable" in Q3.
This could be a source of concern for the IMF, official investors and other foreign 'real money' investors, he adds.
"In our view, the next round of capital account/forex reform measures will likely address this issue, for example by granting onshore banks greater access to the offshore CNH market, and vice-versa," adds the head of research.
In his opinion, policy makers are more likely to focus on the insufficient onshore-offshore arbitrage activity now that the RMB's SDR implementation is soon out of the way.
"The next big step for the RMB is improving the circulation of it between the onshore and offshore markets. It's more about seeking better convergence between CNY and CNH," says Mackel.
Singapore dollar bulls are gathering in the swap market
Who wins or loses if Kim Jong-Un agrees to denuclearise?
Singapore takes split-second chance to be FX hub for Asia's rich
Suntec REIT acquires Adelaide office building for $141 mil
Wilmar's YKA Shenzhen listing to raise visibility of China business
Will Venture Corp take another hit as customer guides for lower growth?
Keppel DC REIT 2Q DPU rises 6% to 1.93 cents on acquisitions
CapitaLand Commercial Trust declares 1.9% higher 2Q DPU to 2.20 cents on new acquisitions


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Zgromadzenie Narodowe (hisz. Asamblea Nacional) – jednoizbowy parlament Wenezueli, główny organ władzy ustawodawczej w tym kraju. Liczba deputowanych nie jest stała, każdy stan oraz stolica wybierają po trzech przedstawicieli. Skład uzupełniany jest na podstawie wyniku uzyskanego przez podzielenie liczby ludności przez 1,1% całej populacji kraju. Pozostałe trzy mandaty zarezerwowane są dla przedstawicieli wenezuelskich plemion, wybieranych oddzielnie przez wszystkich obywateli. W wyborach stosuje się ordynację proporcjonalną. Zgromadzenie Narodowe obraduje w Federalnym Pałacu Legislacyjnym w Caracas.

Kadencja parlamentu trwa 5 lat.

Przypisy

Wenezuela
Polityka Wenezueli


text:
British soldier calls for help from Jeremy Hunt after being sentenced to jail for fighting ISIS
By Samantha King
A former soldier sentenced to jail in Turkey after fighting the Islamic State terror group is "begging" for a firm intervention from the UK Government.
Twenty-five-year-old Joe Robinson from Lancashire, was sentenced to seven-and-a-half years' imprisonment after being accused of fighting alongside the People's Protection Units of Syrian Kurdistan (YPG), which the Turkish state considers a terrorist organisation.
The YPG is not banned in the UK and Mr Robinson claims he only spent a month with them in Syria while providing medical support to civilians.
He is currently on bail in Kusadasi awaiting an appeal and has called on Foreign Secretary Jeremy Hunt to intervene after writing to former foreign secretaries Boris Johnson and Alan Duncan over the case to no avail.
'Emotionally drained'
"It's got to the point now where I'm mentally, physically and emotionally drained," he told ITV News.
"I'm a proud man but I am literally at the point where I am begging, begging the British Government to do something."
A Foreign Office spokeswoman said officials are ready to provide him with further assistance, adding: "We have been following this case very closely and have raised it with the Turkish authorities."
However, in an interview with Julia Hartley-Brewer, Hannah Lucinda-Smith, a correspondent for the Times in Turkey, said: "The British government is trying to keep its relations with Turkey on a good level for all kinds of reasons, for trade reasons, for security reasons. So I think it's quite unlilely we're going to see Jeremy Hunt make any public statements on this case.
"This is the problem for Joe Robinson, Turkey considers the YPG primarily not to be the troops fighting against ISIS as we see them as in the west, but as linked to a terrorist group that they themselves are fighting against."


Erythroid Krüppel-like factor (EKLF or KLF1) is a transcriptional regulator that plays a critical role in lineage-restricted control of gene expression. KLF1 expression and activity are tightly controlled in a temporal and differentiation stage-specific manner. The mechanisms by which KLF1 is regulated encompass a range of biological processes, including control of KLF1 RNA transcription, protein stability, localization, and posttranslational modifications. Intact KLF1 regulation is essential to correctly regulate erythroid function by gene transcription and to maintain hematopoietic lineage homeostasis by ensuring a proper balance of erythroid/megakaryocytic differentiation. In turn, KLF1 regulates erythroid biology by a wide variety of mechanisms, including gene activation and repression by regulation of chromatin configuration, transcriptional initiation and elongation, and localization of gene loci to transcription factories in the nucleus. An extensive series of biochemical, molecular, and genetic analyses has uncovered some of the secrets of its success, and recent studies are highlighted here. These reveal a multilayered set of control mechanisms that enable efficient and specific integration of transcriptional and epigenetic controls and that pave the way for proper lineage commitment and differentiation.
The correct balance of progenitor cell proliferation versus lineage-committed differentiation is central to the homeostasis of the hematopoietic system (1). The isolation and functional evaluation of transcription factors that direct expression of lineage-restricted target genes have led to significant advances in understanding how intracellular control of lineage commitment occurs at the molecular level (2). A major unresolved issue is the mechanism of how these proteins interact with chromatin-modifying factors and with the basal transcription machinery to direct the genotypic and phenotypic changes manifested during the process of lineage differentiation.
Erythroid Krüppel-like factor (EKLF) is a red cell-enriched DNA binding protein that interacts with its cognate 5′-CCMCRCCCN-3′ element at target promoters and enhancers (3) and is the founding member (now named KLF1) of the KLF family of transcription factors. This family is defined by the presence of three similar C2H2 zinc fingers at the C terminus that comprise its DNA binding domain (3–5). KLF1's role in β-like globin gene regulation has been the major focus of genetic, biochemical, and molecular studies since its discovery (6, 7). KLF1 is absolutely critical for the proper function and development of the erythroid lineage as supported by gene ablation studies in mice, of which the most obvious phenotypic effect is a profound, lethal β-thalassemia at the time of the switch to adult β-globin expression (8–10).
During the last 3 years, KLF1 has more fully emerged as one of the central players in establishing and maintaining global erythroid control (11–13). Recent studies utilizing chromatin immunoprecipitation (ChIP) followed by massively parallel sequencing (ChIP-seq) have demonstrated that KLF1 is physically bound to a large number of promoters, introns, and intergenic regions associated with erythroid cell-specific genes in both progenitors and terminally differentiating erythroid cells (13, 14). Together with expression profiling studies, KLF1's activation target repertoire has expanded beyond the classical β-globin gene to include protein-stabilizing, heme biosynthetic pathway, red cell membrane proteins, cell cycle, and transcription factor genes in both primitive and definitive erythroid cells (12–21). KLF1 activation targets have direct effects on erythroid biology, especially with respect to megakaryocyte-erythroid lineage decisions, control of cell cycle gene expression patterns (e.g., E2f2, E2f4, p18, and p21), and coordination of related events at late stages of erythroid differentiation (reviewed in reference 12).
The multiple defects in erythropoiesis and the erythroid lineage decisions that arise in KLF1-deficient embryos underline the importance of KLF1 in red cell biology. Its multifunctional roles in globin switching, lineage determination, terminal maturation, and human disease have been recently reviewed (12, 13). However, in addition to studies directed at understanding the role of KLF1 in the context of organismal biology, an extensive series of analyses has been performed to decipher the molecular mechanisms that govern KLF1 expression and function. These mechanistic aspects were last summarized more than 10 years ago (6, 7). In the present review, we update the latter studies and focus on recent exciting developments that elucidate KLF1's ability to directly integrate basal transcription and chromatin-modifying machineries, leading to temporal and lineage-restricted transcription of target genes.
Transcription initiation and promoter structure.KLF1 interacts with the p62 subunit of TFIIH (22) and with TAF9 (23), each of which are components of the basal transcription machinery. These interactions are necessary for the optimal transactivation function of KLF1 and lead to two important observations that illuminate functional aspects of KLF1 initiation control. First, the interaction with p62/TFIIH imparts a helix-like structure to the normally unstructured region of the KLF1 activation domain, enabling the prediction and verification of a critical tryptophan amino acid within KLF1 (W73) required for its optimal activation function at the β-globin promoter (22). Since other proteins have been shown to interact with the structurally well characterized zinc finger region of KLF1 (see below), this result suggests that the whole of KLF1 may exist in a tightly structured conformation when complexed with transcription initiation components. Second, the discovery that association between KLF1 and TAF9 (a TATA-binding protein [TBP]-associated protein that is recruited by KLF1 to the β-globin promoter and is necessary for its activation) is not required at all KLF1 activation target promoters (23) suggests that interactions between KLF1 and the basal transcription machinery can be fine-tuned in a manner that may depend on specific promoter architectures and sequences (Fig. 1). In this instance, a conserved downstream promoter element (DPE) (24, 25), located at the β-globin promoter, interacts with TAF9. However, a TAF9-independent mode of KLF1 activation is seen at the alpha-hemoglobin stabilizing protein (AHSP) gene, which does not contain a discernible DPE. Together, these interactions with general transcription components are of critical importance for β-globin expression, since mutation within TFIIH leads to β-thalassemia (26), and some β-thalassemia mutations map to the β-globin DPE (27, 28).
Nonequivalent KLF1 target gene regulatory mechanisms. KLF1 interaction with associated factors is either not required at all promoters (TAF9 ) or leads to differing transcriptional effects (PIAS3 [Siatecka and Bieker, unpublished]). Part of the TAF9 requirement may be related to whether INI and DPE promoter elements are present (23). Nan-EKLF contains a mutation within its second zinc finger (E339D) and recognizes a subset of normal KLF1 cognate elements: class I elements are recognized by both WT and Nan proteins, but class II elements are recognized only by the WT (29). Haploinsufficiency of KLF1 expression leads to a range of effects only on selected target genes (32, 126).
The nonequivalence of KLF1 gene targets (Fig. 1) is also manifested by analyses of the Nan-KLF1 (E339D mutant) interactions with DNA, where "class I" (p21 and AHSP) versus "class II" (KLF3, E2F2, and dematin) promoters are differentially activated compared to the wild type (WT) (12, 29). Additionally, some KLF1 activation targets are repressed (β-globin and AHSP) while others are enhanced (p18, p21, and BKLF/KLF3) by a SUMO-independent interaction with PIAS proteins (M. Siatecka and J. J. Bieker, unpublished). Finally, it must be noted in this context that some genes are uniquely sensitive to haploinsufficient levels of KLF1 (Lu and Bcl11a genes), leading to altered genetic expression patterns and hematologic parameters in humans [In(Lu) and hereditary persistence of fetal hemoglobin [HPFH] (11, 12, 30–33).
KLF1 displays selectivity in its interaction with histones and associates only with histone H3 (34). In this case, the KLF1 zinc finger region interacts with the H3 amino-terminal tail, but this interaction does not enable any discrimination between the H3.1 and H3.3 variants. As a result, the mechanism by which the H3.3 (but not the H3.1) variant is enriched at the β-globin promoter appears more complex than merely discrimination and recruitment by KLF1. However, interaction between KLF1 and H3.3 chaperones may provide a means to establish this enrichment (S. Soni and J. J. Bieker, unpublished).
GATA1 can physically interact with KLF1 and exhibits functional synergy with KLF1 at erythroid promoters as tested by cotransfection (35, 36). It is not known if the physical interaction between GATA1 and KLF1 is required for their synergism. One genome-wide KLF1 study has shown that most of its target sites are located a nonrandom distance away from a GATA binding motif (21). Another study that compared GATA1, SCL, and KLF1 ChIP-seq data suggests there are a significant number of target genes cooccupied by these three factors, forming a transcriptional core network (37). These observations have been supported and further buttressed by demonstrating their selective overlap with sites to which P300 is bound (38). On the other hand, another genome-wide study using the hemagglutinin (HA)-EKLF knock-in mouse (39) showed only a small overlap between KLF1 and GATA- or SCL-binding motifs (14), a difference possibly related to the use of the HA antibody and detection of a greater number of targets. Several recent studies, aimed at investigating interactions between erythroid cell-specific transcription factors and other proteins, have not shown KLF1 in complex with GATA1 in vivo (40–42). Nevertheless, identification of consensus sequences contained within KLF1-bound genomic regions implicates other transcription factors that may partner with KLF1 (21). Identifying these factors and deciphering the mode by which they interact with KLF1 to regulate target gene expression remains an open issue, one whose resolution will illuminate the transcription factor hierarchy (43) that operates to prime and maintain the erythroid program.
Chromatin protein interactions/higher-order structure.KLF1 also interacts with chromatin-modifying and -remodeling factors, such as P/CAF, CBP/p300 (44), and the SWI/SNF complex (45, 46), the latter likely through its interaction with BRG1 (47–50) and possibly BAF47/BAF155 (51). Erythroid cells that lack KLF1 exhibit an aberrant chromatin configuration and altered components at KLF1 target promoters, resulting in histone hypoacetylation and loss of DNase hypersensitivity and an absence of CBP, BRG1, TBP, and RNA polymerase II (Pol II) (52).
KLF1 is necessary for transcription of AHSP mRNA and directly binds to a CACC site in the AHSP promoter (53, 54). In the absence of KLF1, a DNase I-hypersensitive site that is typically present in the promoter is lost, leading to a decrease but not complete loss of acetylated H3 and H4 across most of the AHSP gene, corresponding with loss of expression. KLF1 also directly regulates the erythroid cell-specific Alad(1b) promoter, enhancing the recruitment of other transcription factors, along with BRG1, P300/CBP, and RNA Pol II, that lead to induced changes in the local histone modification and chromatin structure (48). The absence of KLF1 disrupts hypersensitive-site formation at the β-globin (55) and E2F2 (19) genes, suggesting that in general, KLF1 plays a role in regulating chromatin conformation of its target loci. The critical role of KLF1 for these events has been most directly shown at the β-like globin locus, where KLF1 is necessary for formation of the active chromatin hub (ACH), a 3-dimensional nuclear interactome that brings distant DNA sequences into a spatially cooccupying complex (56–58). KLF1 not only binds the proximal adult β-globin promoter in vivo but also interacts with specific regions of the far-upstream locus control region (LCR) (59–61). It is noteworthy that an incomplete chromatin scaffold is formed in the absence of KLF1 (56). Although intergenic transcripts are normally generated throughout the β-locus, KLF1 binding and the three-dimensional (3D) structure of the locus are still retained when transcription initiation or elongation is inhibited (57, 62), suggesting that KLF1 directly drives its formation even in the absence of transcription.
Given the role of KLF1 in indirect regulation of embryonic and fetal globin genes via Bcl11a (30), it is perhaps surprising that KLF1 has been implicated in their direct control (61). In primitive erythroid cells isolated from embryonic day 10.5 (E10.5) murine embryos, the presence of KLF1 is required for enrichment of H3K9Ac (associated with an open chromatin configuration) at HS2 of the β-globin locus, as well as at the Ey and βh1 genes. It is also required for the enrichment of H3K4me3 (associated with active transcription) at HS2 and HS3, in addition to the Ey, βh1, and βmaj genes, despite the absence of βmaj transcription at this point (63). Part of KLF1's control mechanism in primitive cells appears to be coordinately regulated with KLF2 (64). For example, a global comparative analysis of transcripts expressed in WT, KLF1-null, or KLF1/KLF2-null primitive red cells led to the discovery that these two closely related transcription factors synergize to directly regulate c-Myc expression, leading to a network of genes that control the primitive red cell maturation program (65).
In erythroid progenitors, KLF1 is bound to an important enhancer element in the c-Myb promoter that, in conjunction with the LDB1 complex, is part of the ACH that forms at the c-Myb gene. As proposed for the β-like globin locus, the c-Myb ACH is suggested to be involved in transitioning the transcription machinery from initiation to elongation. However, this is a dynamic process: as erythroid cells terminally differentiate, KLF1 and the other proteins that form the ACH dissociate from the complex, and c-Myb expression decreases (66). Together with the effects at the globin locus, these data suggest that the presence of KLF1 is critical for transcription initiation in facilitating the formation of an open chromatin configuration at target loci. These data raise the possibility that in addition to transcription initiation, KLF1 also plays a role in regulating transcription elongation in erythroid cells (67), since the interaction between the upstream LCR and the proximal β-globin promoter, whose 3D chromatin structure requires KLF1 (56, 58), aids in establishing a postinitiation transcription elongation complex (68, 69). KLF1 is also recruited to the α-globin promoter and one of its upstream enhancer elements at late stages of differentiation (60, 70), possibly aiding in intrachromosomal interactions, preinitiation complex recruitment, and the transition from a poised to an elongating transcriptional state (70). However, it remains to be seen if stage-specific regulation of transcription elongation through chromatin looping is a generalized mechanism by which KLF1 functions. An additional detail in this context is the observation that KLF1 interacts with Ppm1b (42); although part of its role relates to KLF1 stability, a tantalizing layer arises from suggestions that Ppm1b dephosphorylates an important residue in the Cdk9 component of the pTEFb elongation complex (71, 72).
Collectively, these data show that KLF1 is an integrator of basic transcription and chromosomal remodeling signals that lead to modified histones, open chromatin, looped domains, and activated transcription. In this context, it is of interest that the majority of in vivo KLF1 binding sites are located at large distances (>10 kb) from transcription initiation sites (21), consistent with long-distance regulation. Use of the HA-tagged KLF1 mouse (39) has yielded slightly different estimates of their relative proportion (14). In any case, as observed with other erythroid factors, only ∼0.1 to 0.5% of theoretical target sites are actually bound in vivo. These interactions play a crucial role in establishing a limited number of transcription factories in vivo that enable efficient coordinate expression of select KLF1 target genes (73). KLF1-associated transcription factories tend to colocalize with loci that are highly transcribed in erythroid cells. For example, the globin genes associate with other active genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes that are larger than transcription factories without KLF1 (74) and that may include PML bodies and Sc35 (75). Such interactions likely go hand in hand with the KLF1-, GATA1- and FOG1-dependent extrusion of the β-globin locus away from the nuclear periphery (76), an event that is necessary for its transcriptional activation (77). The KLF1 protein in erythroid cells from the HA-EKLF knock-in mouse is localized at the periphery in progenitor cells but then is dispersed throughout the nucleus in erythroblasts (14). These data suggest that KLF1 plays a global role in coordinating maximal transcription rates of select erythroid cell-specific genes by regulating their localization to specialized subnuclear compartments. The molecular mechanism by which these precise factories are established and mobilized within the intricate milieu of the nucleus remains a challenging cell biology and biochemical issue.
KLF1 activates a large number of genes that are broadly classified as important for erythroid function and development. It is highly expressed in the megakaryocyte-erythroid progenitor in addition to early differentiating primitive and definitive erythroid cells, and is especially critical at late stages of differentiation (e.g., erythroid lineage induction module 889 ). And yet its direct targets differ in different cell types, for example when comparing erythroid progenitors versus its erythroblast progeny (14) or primitive versus definitive red cells (61, 63). KLF1 is posttranslationally modified by phosphorylation (78), ubiquitylation (79), acetylation (80), and sumoylation (81). Posttranslational modifications of transcription factors can provide alternate protein interaction platforms that lead to varied downstream effects. As a result, these modifications may play a determining role in modulating KLF1's interactions with other proteins, fine-tuning its function in erythroid precursor, progenitor, and mature cells at different differentiation stages (Fig. 2).
Summary of KLF1 posttranslational modifications and consequent protein-protein interactions. (Top) Schematic diagram of KLF1, showing the general locations of phosphorylation, sumoylation, acetylation, and ubiquitinylation modifications and their effects on subsequent protein-protein interactions and transcriptional readout. Those that are essential for optimal function as an activator are in green; those for repression are in pink (based on references 78, 79, 81, 82, and 44). Functional regions are based on mouse amino acid numbering (3, 79, 95, 127). (Bottom) Tabulation of KLF1 protein interactions, their interaction sites and any subsequent modification, and their downstream effects on transcription/chromatin. m, mouse; h, human.
Acetylation of KLF1 by CBP or P300 occurs at two sites, K288 and K302, and individually these play a critical role in subsequent protein-protein interactions that lead to different functional effects. Directed mutagenesis of K288 (but not K302) decreases the ability of KLF1 to transactivate the β-globin promoter and renders it unable to be superactivated by coexpressed p300 or CBP (44). Acetylated KLF1 has a higher affinity for the SWI/SNF chromatin remodeling complex and is a more potent transcriptional activator of chromatin-assembled templates in vitro (44). Using an in vivo rescue system (34), K288 acetylation has been shown to be critical for recruitment of CBP to the β-globin locus, modification of histone H3, occupancy by KLF1, opening of the chromatin structure, and transcription of adult β-globin (34).
On the other hand, the interaction of KLF1 with Sin3a is dependent on the acetylation status of KLF1 at K302. Mutagenesis of Lys-302 renders KLF1 no longer able to bind Sin3A, and it loses most of its repression activity (82). Endogenous KLF1 preferentially interacts with Sin3a in primitive erythroid cells and to a lesser extent in definitive erythroid cells (82, 83), suggesting that there are differentiation stage-dependent differences in KLF1 repression activity, possibly arising from variations in acetylation status (82). The biological significance of KLF1-mediated gene repression via this mechanism remains to be resolved and is further complicated by observations that an intact zinc finger region and thus target DNA specificity is not required for Sin3a interaction or repression.
KLF1 is also posttranslationally modified by SUMO (81) at a unique amino acid residue, K74, with the aid of PIAS1 as its E3 ligase. This leads to a more efficient interaction with the Mi2β subunit of the NuRD complex (an interaction that has been observed globally ) and transcriptional repression. Most intriguingly, a significant consequence of mutation in the KLF1 SUMO acceptor site is the loss of its inhibitory effect on megakaryocytic differentiation. The role of KLF1 in bipotential decisions emanating from the megakaryocyte/erythroid progenitor following gain- and loss-of-function experiments has been reviewed elsewhere (12). The importance of sumoylation in this process was shown in two ways. Using K562 cells, it was found that induction of megakaryopoiesis (with tetradecanoyl phorbol acetate [TPA]) was inhibited by the presence of wild-type KLF1 compared to that seen in the presence of KLF1 with the K74R mutation (K74R-KLF1), which exhibited little inhibition of the process. This was further supported by in vivo tests, where transgenic misexpression of WT KLF1 in megakaryocytes effectively decreased megakaryocyte cellularity and colony formation in the bone marrow, whereas transgenic bone marrow from the K74R-KLF1 line revealed no effect of the misexpression. Notably, erythroid colony formation by either of the transgenic lines, compared to the nontransgenic line, was not significantly affected. As a result, the availability of the SUMO modification site in KLF1 is absolutely critical for it to exert its normal inhibition of megakaryopoiesis prior to red cell onset (81). This KLF1 status may be directly relevant to cross-antagonistic models with Fli-1, a critical megakaryocytic transcription factor whose levels and activity are thought to be directly and/or indirectly repressed by KLF1 (reviewed in references 85 and 12).
At this point, the function of KLF1 as a repressor remains an area of speculation as its direct in vivo interaction with postulated repressed genes has not been extensively characterized. Comparison of expression profiling and ChIP experiments suggest that a majority of KLF1 targets are activated (14, 17, 21, 38); however, the smaller number of putative repressed targets may still be highly biologically significant. A potential complication is that BKLF/KLF3 and KLF8 are downstream targets of KLF1 and interact with a similar target DNA site (16, 86). KLF3 is a transcription factor that functions mainly as a repressor, and a recent model suggests that KLF3 acts as a feedback repressor to temper KLF1 activation of select targets (either promiscuous or specific) at late stages of erythropoiesis (87).
KLF1 is phosphorylated in vivo at serine and threonine residues, and casein kinase II (CKII)-dependent phosphorylation at T41 is essential for a GAL/EKLF(20–124) fusion to behave as an activator of transcription (78). Paradoxically, this study also showed that certain KLF1 kinases in MEL lysates decrease in activity when MEL cells are differentiated with hexamethylene bisacetimide (HMBA), raising the possibility that inhibition of KLF1 phosphorylation or an increase in phosphatase activity also plays a role in erythroid differentiation. Ppm1b, a serine/threonine phosphatase that interacts with KLF1, can inhibit the induction of erythropoiesis, while at the same time it has the ability to stabilize KLF1, augmenting its ability to activate transcription in committed erythroid cells (42). While exact mechanisms for how CKII and Ppm1b regulate erythroid homeostasis are not known and remain open areas of inquiry, these data suggest that both may play a role in part by regulating the transactivation capability and stability of the KLF1 protein. The functional coupling of phosphorylation to acetylation and to sumoylation (88) underscores the need to address whether there is any cross-regulation between these KLF1 modifications.
The mouse mutation Nan (neonatal anemia) was mapped to a single amino acid change (E339D) within the second zinc finger of KLF1 (29, 89, 90). The mutation alters the DNA binding specificity of KLF1 such that it no longer binds promoters of a subset of its DNA targets (29). Remarkably, even when mutant Nan and wild-type KLF1 alleles are expressed at equivalent levels, the mutant form selectively interferes with expression of KLF1 target genes whose promoter elements it no longer binds. This yields a distorted genetic output and selective protein deficiencies that differ from those seen in KLF1-heterozygous and KLF1-null red cells (29). This murine mutation has converged with human disease, particularly a subtype of congenital dyserythropoietic anemia (12, 91–94), where the same amino acid is altered (albeit to lysine; E325K) in human KLF1.
While it may not be surprising that zinc finger mutations that alter KLF1 interaction with its target site lead to functional changes, it should be kept in mind that this region is also responsible for protein interactions (Fig. 2). In some cases, an intact finger structure is not required (e.g., Sin3a  and importins ).
Although most human KLF1 mutations to date encode truncated proteins that ultimately lead to haploinsufficient expression levels, a significant number lead to missense mutations that result in expression of an altered protein (12, 33). It has been noted that most of these are localized to the DNA binding region, particularly the second zinc finger (31, 33, 94, 96, 97), and new mutations continue to support this initial observation (http://lovd.bx.psu.edu/variants.php?action=search_unique&select_db=KLF1). The structural implications of these alterations have been discussed elsewhere (32, 94), but it is interesting to note that, upon inspection of a model of KLF1 bound to its cognate DNA, almost all fall within the side of the protein that forms the interface with DNA (e.g., hE325) (Fig. 3). In most cases, these mutations alter important conserved and direct DNA-interacting amino acids, but a few are at sites that are predicted (from the model ) to be on a surface that faces away from DNA. As discussed above, numerous proteins interact with KLF1 via its zinc finger domain (Fig. 2). One can predict that mutations will be discovered that do not alter the DNA binding activity of KLF1 but disrupt protein-protein interactions, much like acetylated mK302 (hK288) enabling KLF1's interaction with Sin3a (82). In this context, human T334 (hT334) (mouse T348 [mT348]) (96) may fit with this idea and might be affecting an important protein interaction (Fig. 3). Alternatively, changes within amino acids that help orient KLF1 on its target DNA sites may alter the overall structure of dynamics of the fingers, thus indirectly disrupting KLF1's affinity for another associated protein(s). Future structural assessment of tripartite KLF1/protein/DNA complexes will help sort out how the zinc finger motif can assimilate such a mix.
Location of selected amino acids within the KLF1 zinc fingers. The KLF1 structural model is based on the Zif268-DNA complex (98). Note that human (h) and mouse (m) numberings are not equivalent. KLF1/DNA structure is viewed at different angles (A, B, and C). Critical basic amino acids that interact with guanine residues on one strand of its double-stranded DNA (dsDNA) recognition element are selectively highlighted: blue for the first finger (mK306 and mH309), magenta for the second finger (mR336 and mR342), and purple for the third finger (mR364 and mH367). Note the two residues highlighted in green: mK302, known to be acetylated and critical for KLF1 interaction with Sin3a, and hT334, one of a collection of human KLF1 mutations. These are highlighted to demonstrate that they face away from the DNA interaction surface and are not directly involved in protein-DNA interactions. Most human KLF1 missense mutations are located at amino acids that are critical for its interaction/recognition of its cognate DNA element and face the DNA, such as hE325 (highlighted in red). Mutations are more fully discussed in the text. Images were generated using the MacPyMOL software program.
Transcriptional regulation of KLF1.KLF1 expression is dramatically restricted during early mouse development, a property quite unlike findings for other members of the family but one that effectively circumscribes its functional effects (discussed in reference 99). KLF1 mRNA first appears at the neural plate stage (∼E7.5), where it is strictly localized to the earliest morphologically identified erythroid cells in the blood islands of the yolk sac, followed by a switch to the fetal liver by E10.5 (100). Expression of KLF1 mRNA in the adult mouse is restricted to the bone marrow and the red pulp of the spleen (3, 100). KLF1 transcript levels dramatically increase during hematopoietic differentiation, both in vivo and in cell culture assays (101–103). This is also reflected in the dynamic changes in histone marks (particularly H3K4/K9/K27/K36 and H4K20) and H2A.Z and RNA Pol II occupancy (101). A 950-bp region, located just upstream of the transcription initiation site, is sufficient to generate erythroid cell-specific expression in transient assays (104). This region exhibits the most significant homology upon a seven-species alignment of 30 kb of surrounding genomic DNA (105) and harbors erythroid-restricted DNase-hypersensitive sites. One of these sites (EHS1) behaves as a very strong enhancer, which in conjunction with the proximal promoter (106–108) accounts for KLF1's tissue-specific expression. The importance of this short region has been verified in vivo by the use of transgenic mice, where the 950-bp piece is sufficient to drive reporter (LacZ) expression specifically to the fetal liver and to the hematopoietic, but not endothelial or visceral endoderm, compartment of the yolk sac (109). Removal of the endogenous EHS1 enhancer decreases KLF1 levels by 50-fold (110).
Mouse embryonic stem (ES) cells differentiating in serum-free medium were used to show that BMP4 is necessary and sufficient to induce KLF1 expression as embryoid bodies (EBs) are being formed (111). The involvement of the Smad pathway in this process was directly implicated by showing that interference by constitutive expression of a dominant-negative BMP receptor or of the inhibitory Smad6 obviates KLF1 expression even in the presence of serum (111), results also seen after manipulation of Smad6 levels in human CD34+ cells (112). Related to this observation, conserved Gata and Smad binding motifs are located precisely within the previously mapped upstream enhancer and proximal promoter sequences, and their importance has been verified by mutational analyses using a KLF1 promoter/green fluorescent protein (GFP) transgene that faithfully recapitulates the onset of endogenous KLF1 expression during mouse EB differentiation (105). ChIP of GATA proteins reveal a switch in their occupancy (113, 114) when comparing early versus late times of EB differentiation, corresponding to GATA2 in the progenitor stage, followed by GATA1 after lineage commitment (105). In addition, use of a doxycycline-inducible short hairpin RNA (shRNA) line directed against Smad5 verified its critical importance for KLF1 expression. Together with the promoter analyses, these data led to the proposition of a two-tiered mechanism for transcriptional regulation of KLF1, with the GATA2 and SMAD5 proteins initially generating low transcript levels, followed by high quantities of KLF1 transcript after the GATA1 protein is produced (105). This partly explains the sustained level of KLF1 expression in GATA1-null erythroid cells (115). Both GATA1 and GATA2 have been shown to bind the KLF1 promoter in vivo (116, 117), and GATA2/SMAD4 (the partner of SMAD5) form part of a combinatorial interaction network (51). In addition, KLF1 retains a GATA1 "bookmark" that enables its rapid reactivation after mitosis (118).
As predicted from studies showing that KLF1 is highly expressed in the common megakaryocyte/erythroid progenitor (102), GFP+ fetal liver cells from a KLF1 promoter/GFP reporter mouse are enriched for erythroid and megakaryocyte progenitors as tested by colony-forming assays (105). This is also true when similar assays are performed with EB-derived cells.
Other proteins are also critical for KLF1 expression. Part of the mechanism by which c-Myb promotes erythropoiesis and represses megakaryopoiesis is by in vivo binding and transactivation of human KLF1, likely within the upstream enhancer region (119). It is probable that c-Myb plays a critical role in onset of KLF1 transcription specifically in progenitors. Analysis of the Gata1-binding region of the KLF1 promoter shows occupancy by the SCL, E2A, HEB, Zbtb7a/LRF/Pokemon, Fog1, and Mi2β proteins (117). How the proper protein complexes form at the upstream enhancer and proximal promoter regions that together are required for tissue-specific expression remains unanswered; its resolution will be relevant to understanding early events in the developing yolk sac as well as during hematopoiesis, when KLF1 is first detected in the multipotent and common myeloid progenitors (102).
Regulation of KLF1 stability.The expression of the KLF1 protein is also regulated by proteosomal control of its stability (79). KLF1 can be ubiquitinated on multiple lysines that span its primary sequence, thus excluding sumoylation or acetylation from playing a controlling role. Although its two conserved PEST sequences are not required for ubiquitination, they may serve as docking sites for proteasome-mediated unfolding of the KLF1 protein prior to degradation (79). The Ppm1b protein phosphatase interacts with the KLF1 PEST1 sequence and increases the stability of KLF1 via its zinc finger domain, thereby regulating the availability of KLF1 to activate transcription (42).
The requirement for tight regulation of KLF1 levels is underlined by evidence that KLF1 haploinsufficiency perturbs regulation of erythropoiesis (reviewed in references 31, 12, and 33). Primitive erythroid cells in KLF1+/− mice possess ruffled membranes and exhibit an abnormal display of cell surface proteins, including an ectopic display of megakaryocytic cell surface markers (18). Haploinsufficiency of human KLF1 is linked to decreased levels of the Lu antigen in red cells (32) and to aberrant regulation of globin expression in adults; some patients that are borderline HbA2 or exhibit a hereditary persistence of fetal hemoglobin (HPFH) are heterozygous carriers of a nonsense mutation that ablates one KLF1 allele (12, 31, 33).
Last, KLF1 protein levels in definitive erythroid cells are 3-fold that of primitive erythroid cells (observed in references 63 and 61, although not in reference 120), possibly leading to altered binding patterns seen at different globin promoters. These observations highlight the idea that controlling the level of KLF1 gene transcription and stability of the protein likely play important roles in regulating its protein-protein interactions and subsequent involvement in downstream processes.
KLF1 possesses two nuclear localization signals (NLSs) near the carboxyl end that individually contribute to maximal nuclear localization (95, 121). The first NLS spans amino acids (aa) 275 to 296 of murine KLF1, a region adjacent to the second NLS that overlaps the KLF1 zinc fingers (aa 293 to 376). The acetylation status of K288 in NLS1 is irrelevant to its activity. KLF1 zinc fingers bind well in vitro to Rch-1 (importin α2) and importin β (95). Heterokaryon assays demonstrate that KLF1 is able to shuttle out of the nucleus, although its nuclear reentry is rapid (122). KLF1 does not contain an obvious nuclear export signal, and shuttling is relatively insensitive to leptomycin B inhibition of CRM1-mediated nuclear export.
In spite of the existence of two domains that behave as strong nuclear localization signals, a significant proportion of KLF1 quite unexpectedly resides in the cytoplasm (14, 73, 75, 122). Immunofluorescent and biochemical assays show that a substantial proportion of endogenous KLF1 is cytoplasmic at steady state in all erythroid cells examined (primary and MEL), that this pattern does not appear to change during either erythroid development or terminal differentiation, and that its nuclear/cytoplasmic ratio is not altered by a variety of treatments (122). Intriguingly, the murine KLF1 protein displays subtle yet distinct biochemical (migration on an SDS-PAGE gel) and functional (altered electrophoretic mobility shift assay [EMSA] complex) differences depending on which subcellular compartment it is isolated from (122), with PEST sequences (79) possibly playing a role in these differences.
The biological significance of KLF1 differential localization is unclear. Each KLF1 population may undergo different posttranslational modifications by virtue of being in different subcellular compartments, possibly enabling its differential retention. Such a mechanism may account for changes in DNA binding and transcription activity of KLF1 over the course of erythroid differentiation. Whether it is sequestered in a latent form or whether it maintains a totally separate function in the cytoplasm remains to be established.
Questions related to mechanistic details that remain outstanding have been alluded to throughout this review. In addition, other recent observations point to new unexplored areas of interest. For example, KLF1's critical role at late stages of erythropoiesis is further supported by the observation that it is expressed in the central macrophage of the erythroblastic island, where it activates DNase IIα (123), a protein that aids in the digestion of nuclei extruded in erythroblastic islands during erythroid enucleation (124). A totally unexpected observation is that an alternatively spliced form is found in the testis (125). Yet another interesting facet is that (paradoxically) both pro- and antiapoptosis pathway components targeted by KLF1 are enriched (13). Analysis of their activities in KLF1-null cells indicates that apoptosis is indeed perturbed but not in a straightforward way (38). Finally, it is likely that non-mRNA-coding genes, such as those encoding microRNAs (miRNAs), are also regulated by KLF1. In this regard, it is of interest that two recently identified long noncoding RNAs (lncRNAs) that exhibit erythroid cell-specific expression are directly regulated by KLF1 (38).
Recent studies from our laboratory have been supported by PHS grants R01 DK046865, R01 DK077822, and RC1 DK086200 and by NYSTEM contract CO26435.
We thank Jim Omichinski and Shefali Soni for comments on the manuscript.
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De Australische Arbeiderspartij (ALP, vaak ook kortweg als Labor aangeduid) is de oudste politieke partij van Australië. Het is een sociaaldemocratische partij die officieel verbonden is met de vakbonden. Sinds 2013 zit de partij weer in de oppositie.

Geschiedenis
Er is geen exacte datum bekend waarop de ALP gesticht zou zijn, omdat ze afkomt van arbeidersbewegingen uit verschillende regio's van Australië. Dergelijke bewegingen bestonden in Nieuw-Zuid-Wales en Queensland al vanaf ongeveer 1890. De partij was serieus en officieel actief vanaf 1891 in Nieuw-Zuid-Wales, vanaf 1893 in Queensland, en later in de rest van het land. In 1899 vormde Anderson Dawson in Queensland voor het eerst ter wereld een minderheidsregering van de arbeiderspartij. Deze regering heeft het één week volgehouden.

De "Federal Parliamentary Labor Party" had haar eerste vergadering op 8 mei 1901 in het parlementsgebouw in Melbourne. Dit wordt tegenwoordig gezien als de datum waarop de partij gesticht is.

De ALP kende gedurende de beginjaren een explosieve groei in het aantal leden. De eerste landelijke (minderheids)regering werd gevormd in 1904, waarna in 1910 voor het eerst een meerderheid in het parlement werd behaald.

Vanaf de jaren zeventig heeft de partij afstand gedaan van haar socialistische inslag en werd het een sociaaldemocratische partij. In de jaren tachtig was de partij zelfs verantwoordelijk voor zaken als het privatiseren van enkele overheidsdiensten en het dereguleren van veel tot dan toe strak geregelde industrieën. Dit gebeurde daarvoor enkel door conservatieve partijen.

Vanaf het ontstaan tot aan de jaren vijftig waren de ALP en haar vakbonden de grootste voorstanders van de White Australia Policy, die alle niet-Europese immigratie in Australië verbood. Dit beleid kwam voort uit 19de-eeuwse theorieën over "rassenzuiverheid" en deels ook uit de angst voor concurrentie op de arbeidsmarkt. In de praktijk was de partij tegen iedere vorm van immigratie. Dit duurde voort tot na de Tweede Wereldoorlog, toen de regering een groot immigratieproject startte. Tegenwoordig is de partij groot voorstander van multiculturalisme, hoewel sommigen in de partij zich nog wel tegen grote aantallen immigranten keren.

Ideologie

Het beleid van de Australian Labor Party wordt iedere drie jaar door de Labor's National Conference bepaald. Het ALP-beleid rond verkiezingstijd wordt bepaald door het kabinet (als de partij op dat moment de regeringspartij is) of door het schaduwkabinet (als ze in de oppositie zitten).

De ALP wordt over het algemeen gezien als een sociaaldemocratische partij, maar in werkelijkheid heeft de partij geen officiële ideologie. De partij is opgericht door vakbondslieden, en staat in principe nog steeds onder hun leiding. Het partijbeleid staat altijd wel tot op zekere hoogte in het teken van de belangen van de vakbonden. Bekeken over de geschiedenis zie je daarom hoe de ALP bijvoorbeeld heeft gepleit voor zowel tariefsverhogingen als -verlagingen, voor White Australia Policy en multiculturalisme, voor nationalisatie en privatisering, en voor isolationisme en internationalisme.

Hoewel het nooit een socialistische partij is geweest, zijn er wel altijd een aantal overtuigde socialisten actief geweest binnen de ALP. Na de Eerste Wereldoorlog en de Russische Revolutie van 1917, groeide binnen de vakbonden de steun voor het socialisme, en op het jaarlijkse vakbondsoverleg in 1921 werd een voorstel aangenomen tot het "socialiseren van industrie, productie, distributie en handel." Dit leidde ertoe dat de ALP in 1922 een soortgelijk "socialistisch streven" ondertekende. Dit bepaalde het beleid gedurende vele jaren.

Hoewel het beleid van Labor tijdens haar regeringsperiode 1983 - 1996 vaak neoliberaal genoemd werd (bijvoorbeeld privatisering en ruime tariefsverlagingen), keert men zich sinds 1996 tegen het beleid van de huidige regering van John Howard. Men neigt eerder weer naar de oorspronkelijke ALP-standpunten op basis van eerlijkheid en gelijkheid wat betreft economie, onderwijs, gezondheidszorg en industrie.

Sinds 2004 heeft de ALP zich vooral gericht tegen Howards deregulering van de arbeidsmarkt en de beperking van de rechten van vakbonden. De partij heeft bij de verkiezingen van 2007 zelfs gezworen dat het terugdraaien van Howards industriewetten hun belangrijkste verkiezingspunt zou worden. Na het winnen van die verkiezingen maakte Labor die belofte meteen waar door het ondertekenen van het Kyoto-protocol.

Facties
De (federale) ALP is een ideologisch erg verdeelde partij en bestaat uit twee geïnstitutionaliseerde facties: Labor Right en Labor Left, die respectievelijk de rechter- en de linkervleugel van de partij vormen.
Labor Right: gematigd economisch liberaal, fiscaal en (gematigd) sociaal conservatief (centrum tot centrum-rechts). Labor Right is voorstander van de zogenaamde Derde Weg, een economische leer die het midden houdt tussen kapitalisme en socialisme. In het verleden was Labor Right sterk anticommunistisch. De factie wordt gesteund door verschillende, meer conservatieve vakbonden (o.a. de katholieke middenstandsvakbond SDA). Oud-premier Kevin Rudd behoort tot de Labor Right-factie.
Labor Left: gemengde economie, fiscaal en sociaal progressief (centrum-links). Labor Left is, anders dan Labor Right openlijk socialistisch.
De meer militante vakbonden en hun leiders steunen deze factie. De factie Oud-premier Julia Gillard behoort tot Labor Left. Dit is niet geval in de parlementen van de deelstaten. In de ene deelstaat domineert Labor Right, in andere deelstaten domineert Labor Left. In het federale parlement houden de twee facties zich qua ledental in evenwicht.

Partijstructuur
De ALP is een democratische, federale partij, met zowel leden als eraan verbonden vakbonden. Onderling beslissen zij over het beleid van de partij en kiezen ze commissies en kandidaten voor publieke functies. De overgrote meerderheid van de Australische vakbonden is verbonden aan de ALP, zodat zij voor een groot deel bijdragen aan de inkomsten ervan. Losse leden betalen ook lidmaatschapsgeld, maar bij hen wordt dat bepaald afhankelijk van hun eigen inkomen. Er zijn ongeveer 50.000 losse leden, alhoewel dit aantal nogal kan veranderen naargelang de partij wel of geen succes heeft bij de verkiezingen.

De partij doet zowel mee aan de landelijke verkiezingen als aan deelstaatverkiezingen en sommige lokale verkiezingen. Op alle niveaus dienen kandidaten een verklaring te onderschrijven waarin ze zweren dat ze altijd zullen stemmen volgens het partijstandpunt. Soms wordt ook van ze verwacht dat ze een deel van hun inkomsten afstaan aan de partijkas.

Trivia
 Hoewel in het Australisch-Engels het woord labour wordt gespeld met de uitgang "-our", heeft de ALP in 1912 gekozen om het uiteinde "-or" te gebruiken. De naamswijziging vond plaats op initiatief van King O'Malley, die uit de Verenigde Staten afkomstig was.

Externe links
 Website Australian Labor Party

Referenties


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Tag Archives: the devil's cinema
Ratings: Dateline's Deadly House of Cards
It looks like Dateline NBC's rerun of "Deadly House of Cards," which featured my narrative non-fiction book on the Mark Twitchell case, has resonated with millions of people.
Preliminary ratings data has placed the two-hour special at the top of the list for Saturday night US broadcasts. And many viewers shared their reactions about the case on social media:
@samcgirl @DatelineNBC Very different from any episode I've ever seen. There's usually twists — but to this extent? Wow…
— Jason (@jfinnell86) July 20, 2014
Some viewers were shocked by the connections between the real-life serial killer investigation and both Hollywood and the Dexter television series.
This episode of #Dateline is mind boggling. I bet a lot of murderers do get ideas from TV shows.
— Nicole (@DarkXTwisty3) July 20, 2014
This is an angle I explored in-depth while researching and writing The Devil's Cinema. Last night, I also shared this excerpt from a prison letter Mark Twitchell sent me, where he responds to my questions about his links with Dexter and his crimes.
2/2 #Dateline Yrs of research, 350pgs of Mark Twitchell's prison letters, etc. Asked about #Dexter. He wrote me this: pic.twitter.com/iYLMkEoknk
— Steve Lillebuen (@lillebuen) July 20, 2014
Mark Twitchell has always denied that he is a murderer. It's clear from this letter that he also seeks to downplay his fascination with Dexter too.
This entry was posted in Crime and Justice, Twitchell and tagged Copycat Killers, Dateline NBC, Deadly House of Cards, Dexter, Dexter Copycat, Mark Twitchell, the devil's cinema, Twitter on July 20, 2014 by Steve.
Dateline NBC: Deadly House of Cards
Wannabe serial killer Mark Twitchell remains a topic of great interest with Dateline NBC revisiting the case in a rerun of "Deadly House of Cards" this Saturday evening.
I was interviewed at length about my research into the case for the two-hour special, but couldn't share any of the exclusive details. Instead, that information all ended up in The Devil's Cinema, my narrative non-fiction account of the entire case and trial.
There have been a few developments since the episode first went to air. No word yet on if this is simply a rebroadcast or if the producers are tacking on an update at the end of the episode.
This entry was posted in Crime and Justice, Twitchell and tagged Dateline NBC, Deadly House of Cards, Dexter killer, Edmonton, Mark Twitchell, Steve Lillebuen, the devil's cinema on July 19, 2014 by Steve.
Ned Kelly Award Shortlist
MELBOURNE — The Devil's Cinema has been shortlisted for the Ned Kelly Award in true crime writing for its portrait of would-be serial killer Mark Twitchell and his "life lived as performance."
The judges of Australia's top crime writing prize have a lot of great things to say about the book:
Lillebuen delves into difficult psychological ground, but manages to portray a life lived as performance. … Well judged and chilling. A highly detailed narrative about a puzzling subject. … The depth of research and analysis results in giving credibility to a story so bizarre it might otherwise be relegated to a work of fiction.
This nomination from the Australian Crime Writers Association comes only a few weeks after the book won Canada's Arthur Ellis Award in non-fiction crime writing.
It's been a dream run to see this story connect with so many readers and critics, both in North America and now across Australia.
This entry was posted in Crime and Justice, Twitchell and tagged Arthur Ellis Award, Dexter killer, Dexter Morgan, Mark Twitchell, Ned Kelly Award, Penguin Australia, Steve Lillebuen, the devil's cinema, True Crime on August 2, 2013 by Steve.