Research Summary: Our study uncovers how pioneer transcription factors gain access to tightly packed heterochromatin regions, initiate transcription from within, and ultimately promote the establishment of gene silencing through heterochromatin assembly.
Author interview: Dr. Manjit Kumar Srivastav is a postdoctoral fellow at the National Cancer Institute, National Institutes of Health. He obtained his Ph.D. from Jawaharlal Nehru University in India. His research interest lies in transcriptional regulation and chromatin biology.
Lab: Dr. Shiv I.S. Grewal, National Institutes of Health (NIH) – National Cancer Institute (NCI), Bethesda, MD, USA.
What was the core problem you aimed to solve with this research?
The genome of eukaryotic cells is packaged into chromatin, a complex of DNA and proteins that can exist in either a relaxed, transcriptionally active form (euchromatin) or a condensed, transcriptionally silent form (heterochromatin). While heterochromatin is essential for maintaining genomic stability and regulating gene expression, it presents a conundrum: it is tightly packed and considered inaccessible to the transcription machinery, yet paradoxically, transcription is required within these regions to establish and maintain their silent state.
This paradox led us to ask a fundamental question: How is transcription initiated in regions that are, by definition, transcriptionally silent and structurally inaccessible? More specifically, what allows the first transcriptional activity that produces RNA molecules necessary to trigger gene silencing in heterochromatin?
Our aim was to unravel this mystery by identifying the molecular actors and mechanisms that enable transcriptional initiation within heterochromatin. Solving this problem not only enhances our understanding of gene regulation but also sheds light on the broader principles of chromatin dynamics, epigenetic inheritance, and developmental biology.
How did you go about solving this problem?
To tackle this question, we used a combination of high-throughput experimental techniques. First, we performed ChIP-Seq to generate genome-wide maps of transcription factor (TF) binding sites. This allowed us to identify which TFs specifically associate with heterochromatic regions. Building on these results, we conducted RNA-Seq analysis to examine how TF binding affects gene expression. By comparing transcriptional profiles, we observed how these factors influence the expression of heterochromatic repeat elements, shedding light on their regulatory roles in initiating transcription within silent chromatin. Finally, we used live-cell fluorescence imaging to directly visualize the biological effects of these TF-derived transcripts. This approach enabled us to monitor how pioneer factor activity impacts heterochromatin formation.
How would you explain your research outcomes (Key findings) to the non-scientific community?
DNA in our cells is tightly packed into a structure called chromatin, which exists in two main forms: euchromatin and heterochromatin. Euchromatin is generally open and accessible, allowing genes to be actively transcribed. In contrast, heterochromatin is tightly compacted and traditionally considered inaccessible to transcription. Our study revealed that a special group of proteins—called pioneer transcription factors—can access these heterochromatic regions. Importantly, they don’t just bind; they initiate transcriptional activity that sets off a cascade of molecular events, ultimately leading to the formation and reinforcement of heterochromatin itself. These factors briefly “unlock” silent regions to produce signals that ensure those same regions remain silent—a finely tuned mechanism critical for cellular identity.
“This work reveals a mechanism by which pioneer transcription factors initiate gene silencing from within transcriptionally silent heterochromatin”.
What are the potential implications of your findings for the field and society?
Our study reveals a fundamental mechanism by which cells regulate gene expression and establish chromatin states, challenging long-standing assumptions about the nature of heterochromatin. Traditionally viewed as a silent and impenetrable region of the genome, heterochromatin is in fact transcribed at specific times, raising the paradox of how transcription is initiated in such tightly compacted domains. By identifying two pioneer transcription factors—PhpCNF-Y, a histone fold-containing protein complex, and Moc3, a zinc-finger protein—that can access these regions and trigger transcription, we provide a critical missing link in understanding how gene silencing programs are first established. This finding expands the known role of pioneer transcription factors, previously recognized for activating genes in heterochromatin, to include the initiation of transcriptional repression from within heterochromatin itself. In doing so, it redefines how we think about transcription factor activity in the context of chromatin accessibility and epigenetic regulation.
Importantly, we show that the initial transcription of heterochromatic repeat elements produces RNA molecules containing cryptic introns. These unusual introns appear to stall the spliceosome, thereby acting as molecular signals to recruit the RNA interference (RNAi) machinery. The resulting small RNAs guide silencing complexes, such as the RITS complex, to specific genomic loci and promote histone H3K9 methylation through the recruitment of the Clr4/Suv39h methyltransferase. This cascade leads to the de novo formation of heterochromatin, demonstrating that transcription itself is not merely tolerated within silent regions but is a prerequisite for establishing long-term silencing.
The implications of this work extend well beyond the mechanistic details of heterochromatin formation. It sheds light on how cells maintain genome stability and proper gene expression during development and differentiation, where silencing of inappropriate genes and repetitive elements is essential for preserving cell identity. The ability of pioneer factors to shape chromatin landscapes during these critical transitions has profound relevance for developmental biology and regenerative medicine. Moreover, dysregulation of chromatin architecture is a hallmark of many human diseases, especially cancer, where inappropriate gene expression or loss of heterochromatin integrity can lead to genomic instability and tumor progression. Understanding how specific transcription factors initiate and maintain heterochromatic silencing offers a new perspective on disease mechanisms and reveals potential molecular targets for therapeutic intervention.
What was the exciting moment during your research?
The moment we saw that transcription factors could bind to and activate transcription in regions of chromatin that were thought to be completely inaccessible was exciting. It felt like uncovering a pathway that nature uses to regulate gene expression. That moment of discovery reminded me why I love doing science: there’s always more to learn, even in areas previously thought to be well understood.
Paper reference: PhpCNF-Y transcription factor infiltrates heterochromatin to generate cryptic intron-containing transcripts crucial for small RNA production. https://www.nature.com/articles /s41467-024-55736-3.
Also read: Protecting heterochromatin from the remodeling machines
