Mechanisms underlying constitutive heterochromatin formation at human centromeres

The National Institute of General Medical Sciences, part of the National Institutes of Health under the Department of Health and Human Services, awarded The Leland Stanford Junior University $152,600 on December 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to investigate mechanisms underlying constitutive heterochromatin formation at human centromeres.

The project will determine how non-catalytic domains of the histone methyltransferase SETDB1 maintain heterochromatin boundaries at centromeres by mutating the triple Tudor domain and putative methyl-CpG binding domain, then evaluating changes in epigenetic profiles of centromere protein A and H3K9me3 using dimelo-seq methodology. The work will also identify genomic loci regulated by interactions between SUV39H1 methyltransferase and RNA by assessing transcription and heterochromatin changes, testing the hypothesis that non-catalytic and RNA-mediated mechanisms target histone methyltransferases to centromeres to initiate and maintain constitutive heterochromatin independently from histone methyltransferase catalytic activity.

Performance takes place in Palo Alto, California, with a period of performance from December 1, 2026, through June 30, 2029.

Abstract

More than half of the human genome consists of repetitive sequences including centromeric repeats which must be silenced by constitutive heterochromatin to ensure genome stability. Constitutive heterochromatin is characterized by TRI-methylation of histone H3 on lysine 9 (H3K9ME3) by three histone methyltransferases (SUV39H1, SUV39H2, SETDB1) and binding of H3K9ME3-marked chromatin by HP1Α. While H3K9 methylation and HP1 protein binding are conserved between fission yeast and humans, the mechanisms that initiate and maintain constitutive heterochromatin are not. To understand how constitutive heterochromatin is regulated in human cells, the proposed project builds upon key findings from our group that point to alternative mechanisms for establishing and maintaining constitutive heterochromatin, independently from histone methyltransferase catalytic activity. We have shown that 1) SETDB1 maintains heterochromatin boundaries at centromeres regardless of its catalytic activity and 2) the direct binding of SUV39H methyltransferases to repeat-derived rnas is required for their localization to pericentromeres. To test the hypothesis that non-catalytic and RNA-mediated mechanisms target histone methyltransferases to centromeres to initiate and maintain constitutive heterochromatin, the proposed project will pursue 3 specific aims. First, this project will determine how non- catalytic domains of SETDB1 contribute to heterochromatin boundary maintenance at centromeres by mutating the triple tudor domain and putative methyl-CPG binding domain of SETDB1 and evaluating changes in the epigenetic profiles of H3 variant centromere protein a (cenp-a) and H3K9ME3 at centromeres using our recently developed dimelo-SEQ method. Second, this project will identify genomic loci regulated by interactions between SUV39H1 and RNA by assessing transcription and heterochromatin changes in SUV39H1/H2 knockout cells and SUV39H1/H2 knockout cells complemented with RNA binding-deficient SUV39H1 using RNA-SEQ and cut & run, respectively. SUV39H1 immunoprecipitation and RNA-SEQ will enable the discovery of rnas that bind to SUV39H1. Third, to discover novel RNA-dependent mechanisms of heterochromatin maintenance, this project will develop a targeted sequencing method for mapping RNA-chromatin contacts associated with specific chromatin proteins. This technique will use antibody-directed capture of rnas associated with specific protein binding sites, followed by proximity ligation or template-switching reverse transcription to generate RNA-DNA linkages. This approach will be used to identify rnas bound to the genome where SUV39H1/H2, SETDB1, and HP1Α act. Results from this project are expected to uncover previously unrecognized, non-catalytic and RNA- mediated mechanisms that regulate constitutive heterochromatin. Given that the transcriptional activation of repeats is associated with several diseases including certain cancers, neurological disorders, and developmental syndromes, a deeper understanding of how constitutive heterochromatin is selectively established and maintained will inform future strategies to target and correct aberrant repeat activation in disease.