Dr. Zhiguo Zhang Research

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Research Summary

Epigenetic inheritance and cancer epigenetics

There are two major research interests and goals in my laboratory: epigenetic inheritance and cancer epigenetics. How epigenetic states are transmitted into daughter cells so called epigenetic inheritance is one of the most challenging, but yet poorly understood, questions in the chromatin and epigenetic fields. In recent years, it has been clear that epigenetic alterations contribute to the development of a variety of diseases including cancer. However, how alterations in epigenetic landscape contribute to tumorigenesis is largely unexplored. Therefore, we are undertaking three major directions to study molecular mechanisms of epigenetic inheritance and cancer epigenetics. We hope that these studies will not only increase our fundamental knowledge about the critical cellular processes, and but will help combat cancer caused by epigenetic alterations.

Major directions/projects in my laboratory

1. Determine how canonical histones H3-H4 are assembled into nucleosomes during S phase of the cell cycle in yeast and human cells

slide1Nucleosome assembly following DNA replication is the “first step” for inheritance of high-order chromatin structure. In general, nucleosome assembly is a stepwise process with deposition of a (H3-H4)2 tetramer first, followed by rapid deposition of two H2A-H2B dimers. We have been addressing how new H3-H4 tetramers are assembled into nucleosomes. Results from my laboratory and others support the following model of nucleosome assembly of new H3-H4 (Figure 1). First, new H3-H4 binds the histone chaperone Asf1 to form the Asf1-H3-H4 heterotrimeric complex. Asf1 then presents H3-H4 to the lysine acetyltransferase Rtt109 for acetylation of H3 lysine 56 (H3K56ac). The Rtt101-Mms1 E3 ligase then preferentially ubiquitylates H3K56ac at three H3 lysine residues (121, 122 and 125) near the Asf1-H3 interface. H3 ubiquitylation weakens the Asf1-H3 interaction and facilitates the transfer of H3-H4 to the histone chaperones CAF-1 and Rtt106, which deposit (H3-H4)2 tetramers onto the DNA for nucleosome formation. While these studies have shed light on nucleosome assembly of new H3-H4, how parental H3-H4 tetramers are assembled into nucleosomes remains largely unknown. In addition, it is largely unexplored how alterations in replication-coupled nucleosome assembly contribute to genome stability and cell fate maintenance. Therefore, we are addressing the following questions related to DNA replication-coupled nucleosome assembly.

  1. How are parental histone H3-H4 tetramers, which carry modifications for inheritance, assembled into nucleosomes?

  2. How alterations in DNA replication-coupled nucleosome assembly contribute to genome instability and cell lineage maintenance of mouse embryonic stem cells?

slide 22. Elucidate how chromatin regulators impact DNA replication using a combination of genetic, biochemical and genomic approaches

DNA replication proceeds with continuous synthesis of leading strand DNA and discontinuous synthesis of lagging strand DNA. However, no reliable method is available to discern whether a protein binds to leading or lagging strands of replication forks. We have designed a method, eSPAN (enrichment and Sequencing Protein-Associated Nascent Strand DNA), that can provide an unprecedented view into the genome-wide association of proteins with leading or lagging strands of DNA replication forks (Figure 2). Using this method, we have shown that PCNA is unloaded from lagging strands of stalled DNA replication forks, revealing novel insight into regulation of DNA replication under replication. Currently, we are also using this method to address the following questions.

  1. How do chromatin regulators including histone modifying enzymes and chromatin remodeling complex impact DNA synthesis of leading and lagging strands?

  2. How is the stability of replication forks maintained under replication stress?

3. Elucidate how mutations in histone H3.3 reprogram epigenetic landscape and promote tumorigenesis

slide 3Histone H3 variant H3.3 differs from canonical histone H3.1/H3.3 by four or five amino acids. H3.1/H3.2 are assembled into nucleosomes during S phase, whereas H3.3 is assembled into nucleosomes by histone chaperone Daxx and HIRA in a replication-independent process. Recent studies have identified histone H3.3 is mutated at high frequency in different tumors. For instance, one allele of H3F3A, one of two genes encoding H3.3, is mutated at lysine 27, replacing lysine methionine, in 60-80% high-grade pediatric brain tumors. In addition, one allele of H3F3B gene, another gene encoding H3.3, is mutated at lysine 36, replacing lysine 36 to methionine, in more than 90% chondroblastoma. Finally, glycine 34 is mutated at high frequency also mutated at high-grade pediatric brain tumors as well as giant cell tumors. How these mutations promote tumorigenesis remains largely unknown. We found that the levels of H3K27 di- and tri-methylation (me2 and me3) are globally reduced in H3.3K27M patient samples. Remarkably, ChIP-seq analyses revealed that H3K27me3, while reduced globally, retains at hundreds of gene loci in H3.3K27M patient cells. Genes retaining H3K27me3 at gene promoters are associated with various cancer pathways (Figure 3). Recently, we have also shown that H3.3K36M mutation dominantly reprograms H3K36 methylation and gene expression in chondroblastomas. These results indicate that expression of H3.3K27M and H3.3K36M mutation from one allele of 16 histone H3 genes, dominantly reprograms the epigenetic landscape and gene expression, which may drive tumorigenesis. Currently, we are addressing the following questions related to impact of histone mutations on tumorigenesis.

  1. How is nucleosome assembly of H3.3 regulated?
    The function of H3.3 in different chromatin context and cellular processes is likely regulated through its assembly. Therefore, exploring the mechanisms whereby nucleosome assembly of H3.3 will help understand how histone H3.3 mutations promote tumorigenesis.

  2. How do different histone mutations promote tumorigenesis?
    We are using CRISPR/Cas9 system to introduce different mutations at different cell types and determine how epigenetic landscapes are altered in different cell types to understand how histone mutations found in cancer cells promote tumorigenesis

We are using CRISPR/Cas9 system to introduce different mutations at different cell types and determine how epigenetic landscapes are altered in different cell types to understand how histone mutations found in cancer cells promote tumorigenesis.