Faculty Bibliography

Exome sequencing is an effective way to identify genetic causes of etiologically heterogeneous conditions such as developmental delay and intellectual disabilities. Using exome sequencing, we have identified four patients with similar phenotypes of developmental delay, intellectual disability, failure to thrive, hypotonia, ataxia, and tooth enamel defects who all have the same de novo R331W missense variant in C-terminal binding protein 1 (CTBP1). CTBP1 is a transcriptional regulator critical for development by coordinating different regulatory pathways. The R331W variant found in these patients is within the C-terminal portion of the PLDLS (Pro-Leu-Asp-Leu-Ser) binding cleft, which is the domain through which CTBP1, interacts with chromatin-modifying enzymes and mediates chromatin-dependent gene repression pathways. This is the first report of mutations within CTBP1 in association with any human disease.

Accurate and sensitive detection of protein-protein and protein-RNA interactions is key to understanding their biological functions. Traditional methods to identify these interactions require cell lysis and biochemical manipulations that exclude cellular compartments that cannot be solubilized under mild conditions. Here, we introduce an in vivo proximity labeling (IPL) technology that employs an affinity tag combined with a photoactivatable probe to label polypeptides and RNAs in the vicinity of a protein of interest in vivo. Using quantitative mass spectrometry and deep sequencing, we show that IPL correctly identifies known protein-protein and protein-RNA interactions in the nucleus of mammalian cells. Thus, IPL provides additional temporal and spatial information for the characterization of biological interactions in vivo.

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 deposition through the deacetylation of H4K16Ac (acetylation of H4K16) and determines the levels of H4K20me2/3 throughout the cell cycle. SirT2 binds and deacetylates PR-Set7 at K90, modulating its chromatin localization. Consistently, SirT2 depletion significantly reduces PR-Set7 chromatin levels, alters the size and number of PR-Set7 foci, and decreases the overall mitotic deposition of H4K20me1. Upon stress, the interaction between SirT2 and PR-Set7 increases along with the H4K20me1 levels, suggesting a novel mitotic checkpoint mechanism. SirT2 loss in mice induces significant defects associated with defective H4K20me1-3 levels. Accordingly, SirT2-deficient animals exhibit genomic instability and chromosomal aberrations and are prone to tumorigenesis. Our studies suggest that the dynamic cross-talk between the environment and the genome during mitosis determines the fate of the subsequent cell cycle.

PR-Set7 is the sole monomethyltransferase responsible for H4K20 monomethylation (H4K20me1) that is the substrate for further methylation by Suv4-20h1/h2. PR-Set7 is required for proper cell cycle progression and is subject to degradation by the CRL4(Cdt2) ubiquitin ligase complex as a function of the cell cycle and DNA damage. This report demonstrates that PR-Set7 is an important downstream effector of CRL4(Cdt2) function during origin of DNA replication licensing, dependent on Suv4-20h1/2 activity. Aberrant rereplication correlates with decreased levels of H4K20me1 and increased levels of H4K20 trimethylation (H4K20me3). Expression of a degradation-resistant PR-Set7 mutant in the mouse embryo that is normally devoid of Suv4-20 does not compromise development or cell cycle progression unless Suv4-20h is coexpressed. PR-Set7 targeting to an artificial locus results in recruitment of the origin recognition complex (ORC) in a manner dependent on Suv4-20h and H4K20me3. Consistent with this, H4K20 methylation status plays a direct role in recruiting ORC through the binding properties of ORC1 and ORCA/LRWD1. Thus, coordinating the status of H4K20 methylation is pivotal for the proper selection of DNA replication origins in higher eukaryotes.

Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone. The associated posttranslational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. We demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, we obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states.

Histone post-translational modifications impact many aspects of chromatin and nuclear function. Histone H4 Lys 20 methylation (H4K20me) has been implicated in regulating diverse processes ranging from the DNA damage response, mitotic condensation, and DNA replication to gene regulation. PR-Set7/Set8/KMT5a is the sole enzyme that catalyzes monomethylation of H4K20 (H4K20me1). It is required for maintenance of all levels of H4K20me, and, importantly, loss of PR-Set7 is catastrophic for the earliest stages of mouse embryonic development. These findings have placed PR-Set7, H4K20me, and proteins that recognize this modification as central nodes of many important pathways. In this review, we discuss the mechanisms required for regulation of PR-Set7 and H4K20me1 levels and attempt to unravel the many functions attributed to these proteins.

The carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) in mammals undergoes extensive posttranslational modification, which is essential for transcriptional initiation and elongation. Here, we show that the CTD of RNAPII is methylated at a single arginine (R1810) by the coactivator-associated arginine methyltransferase 1 (CARM1). Although methylation at R1810 is present on the hyperphosphorylated form of RNAPII in vivo, Ser2 or Ser5 phosphorylation inhibits CARM1 activity toward this site in vitro, suggesting that methylation occurs before transcription initiation. Mutation of R1810 results in the misexpression of a variety of small nuclear RNAs and small nucleolar RNAs, an effect that is also observed in Carm1(-/-) mouse embryo fibroblasts. These results demonstrate that CTD methylation facilitates the expression of select RNAs, perhaps serving to discriminate the RNAPII-associated machinery recruited to distinct gene types

The histone methyltransferase PR-Set7/Set8 is the sole enzyme that catalyzes monomethylation of histone H4 at K20 (H4K20me1). Previous reports document disparate evidence regarding PR-Set7 expression during the cell cycle, the biological relevance of PR-Set7 interaction with PCNA, and its role in the cell. We find that PR-Set7 is indeed undetectable during S phase and instead is detected during late G2, mitosis, and early G1. PR-Set7 is transiently recruited to laser-induced DNA damage sites through its interaction with PCNA, after which 53BP1 is recruited dependent on PR-Set7 catalytic activity. During the DNA damage response, PR-Set7 interaction with PCNA through a specialized 'PIP degron' domain targets it for PCNA-coupled CRL4(Cdt2)-dependent proteolysis. PR-Set7 mutant in its 'PIP degron' is now detectable during S phase, during which the mutant protein accumulates. Outside the chromatin context, Skp2 promotes PR-Set7 degradation as well. These findings demonstrate a stringent spatiotemporal control of PR-Set7 that is essential for preserving the genomic integrity of mammalian cells

Chromatin affects many, if not all aspects, of nuclear organization and function. For this reason, we have focused our attention on elucidating some of the basic mechanisms regulating the formation and maintenance of chromatin, specifically concerning Polycomb repressive complex 2 (PRC2) and PR-Set7. PRC2 is responsible for catalyzing trimethylation of lysine 27 of histone H3 and thus has a critical role in the formation of facultative heterochromatin. PR-Set7 is responsible for catalyzing monomethylation of lysine 20 of histone H4 and is required for proper cell cycle progression and DNA damage response. We have also expanded our work to establish novel techniques and approaches to determine how chromatin is spatially regulated within the nuclear landscape.

The BTB/POZ transcriptional repressor and candidate oncogene BCL6 is frequently misregulated in B-cell lymphomas. The interface through which the BCL6 BTB domain mediates recruitment of the SMRT, NCoR and BCoR corepressors was recently identified. To determine the contribution of this interface to BCL6 transcriptional and biological properties, we generated a peptide that specifically binds BCL6 and blocks corepressor recruitment in vivo. This inhibitor disrupts BCL6-mediated repression and establishment of silenced chromatin, reactivates natural BCL6 target genes, and abrogates BCL6 biological function in B cells. In BCL6-positive lymphoma cells, peptide blockade caused apoptosis and cell cycle arrest. BTB domain peptide inhibitors may constitute a novel therapeutic agent for B-cell lymphomas.