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How Transcription Factor KLF4 Influences Gene Expression

Researchers led by scientists at the Baylor College of Medicine reports the discovery of a mechanism by which transcription factor KLF4 can help to organize chromatin, thus influencing gene expression. The study (“Liquid condensation of reprogramming factor KLF4 with DNA provides a mechanism for chromatin organization”), published in Nature Communications, shows that the binding of KLF4 can cause DNA to condense into a separate liquid phase in a process called biomolecular condensation, which recruits other factors that influence gene expression.

“Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation,” write the investigators.

“Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid–liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site.

“We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.”

“Cells regulate the expression of their genes with transcription factors,” said co-corresponding author Josephine C. Ferreon, PhD, assistant professor of pharmacology and chemical biology and member of the Dan L Duncan Comprehensive Cancer Center at Baylor. “In the current study, we focused on master transcription factor KLF4, which is known to selectively mediate gene expression and reprogramming that determines cell fate.”

Reorganizing chromatin to facilitate gene transcription

Chromatin is a complex, compact, and dense structure made of DNA and proteins. Expressing a particular gene requires that the gene expression molecular machinery has access to that stretch of DNA. Transcription factors such as KLF4 are involved in reorganizing the chromatin to provide access and facilitate gene transcription, but it is not clear how this is accomplished.

By conducting experiments with cells grown in the lab, the researchers discovered that KLF4 forms droplets in the cell nucleus that recruit other transcription factors.

“Imagine mixing oil and water, how they form separate layers, or two liquid phases,” continued Ferreon. “When KLF4 interacts with specific chromatin regions, it forms a condensate that preferentially recruits other molecules that help open the chromatin and mediate gene transcription.”

Other transcription factors participate in biomolecular condensation through unstructured protein regions, but the researchers showed that KLF4 droplets form in cells even if its unstructured regions are not present. Instead, KLF4 droplet formation depends on regions called zinc fingers, which are known to bind DNA.

Single molecule fluorescence experiments show that the three KLF4 zinc fingers, which usually bind in a row to one DNA, can “bridge” between two DNA molecules. “This type of biomolecular condensation involving zinc fingers and DNA has not been seen before,” pointed out Ferreon.

“Formation of this biomolecular condensate is strongly enhanced by a DNA modification called CpG methylation, a change that influences gene expression,” added co-corresponding author Kevin MacKenzie, PhD, associate professor of pathology and immunology and of pharmacology and chemical biology at Baylor. “Our results suggest that the local sequence of DNA and its CpG methylation state enable KLF4 to drive DNA into a separate phase, which helps to organize chromatin in three dimensions.

“Hundreds of human transcription factors contain tandem zinc fingers like those in KLF4, so this class of rapidly evolving proteins may be implicated in chromatin organization through similar ‘bridging’ interactions.”

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