Research

Overview
Eukaryotic chromosomes comprise DNA that is complexed with small basic proteins, histones, and other proteins to generate chromatin. The tight association of these proteins with DNA provides a level of transcription control and contributes to epigenetic mechanisms of gene regulation. In addition to potential effects on chromatin structure, histone modifications act as binding sites/receptors for protein complexes that activate or repress gene transcription. Thus, histone modification can be used to generate a "code" of signals on the surface of the chromosome fiber, which provides regulatory information above that contained in the DNA sequence.

Most of the reactions involved in chromatin modification require metabolites as their cofactors or coenzymes. Therefore, the metabolic status of the cell alters histone modifications and nucleosome structures, impacting epigenetic processes. In addition to dysfunctions of metabolic enzymes, imbalances between metabolism and chromatin activities triggers metabolic disease and changes in lifespan.

Our laboratory focuses on studying the protein complexes that carry out these histone modifications, crosstalk between histone modifications, and protein complexes that recognize the resulting signals. We also search the linkage among transcription, translation, and metabolism through studying the protein complexes directly regulating metabolism in yeast and humans.

1.
H3K36 methylation and the Set2/Rpd3S pathway for restoring chromatin
Chromatin remodeling and modification during transcription elongation is an emerging area of research. Proteins involved in this process include chromatin remodeling complexes (RSC, Chd1, Isw1b) histone chaperones (Spt6, Spt16), a histone methyltransferase (Set2) and a histone deacetylases (Rpd3S). This remodeling during elongation is best understood in yeast, where it seems to promote the passage of RNA polymerase II through nucleosomes and to restore chromatin structure behind the polymerase retaining the original nucleosomal histones (Chromatin resetting). This process is linked to the C-terminal repeats of Pol. II.
RNA polymerase associated Set2 co-transcriptionally methylates H3K36 which is recognized by the Rpd3S HDAC complex leading to deacetylation of nucleosomes in the open reading frame. In the absence of Set2, H3K36 or Rpd3S nucleosomes in transcribed regions become hyperacetylated. We identified the Isw1b nucleosome remodeling complex as another reader of H3K36me3. These and additional results revealed that Set2-mediated K36 methylation had several functions in restoring chromatin structure behind RNA polymerase.  Having worked out the Set2/Rpd3S pathway in yeast we have now turned to study the mammalian Set2 homolog SETD2 (the SET domain-containing methyltransferase). SETD2 non-redundantly trimethylates histone H3 at lysine 36 in mammals. The function of H3K36me3 is linked to DNA methylation, damage repair, and pre-mRNA splicing. However, the knowledge of the regulation of the SETD2 enzyme itself is limited. We want to shed more light on how the enzyme is regulated, with special focus on the role of the functionally uncharacterized N-terminal region and novel binding partners/substrates.
2.
ATP dependent chromatin remodeling complexes and cancer.
SWI/SNF and Cancer Human
SWI/SNF are approximately 2 MDa complexes composed of 12-15 subunits. There are three distinct mature complexes―BAF (BRG1-associated factor), PBAF (polybromo BRG1-associated factor) and ncBAF (non-canonical). These complexes contain one of the two catalytic ATPase subunits, SMARCA4 (BRG1) or SMARCA2 (BRM,) several core subunits including SMARCC1 (BAF155) and SMARCD1, as well as unique subunits such as ARID1A/B for BAF, and PBRM1 and ARID2 specific for PBAF and GLTSCR1 and BRD9 for ncBAF varieties of the complexes. Genes encoding many subunits of SWI/SNF complexes have been found to be recurrently mutated in 20% of all human cancers. Multiple subunits are mutated in a wide spectrum of cancers, emphasizing that individual subunits are important in varying cell types and lineages. Hence an understanding of the mechanism by which SWI/SNF regulates transcription and the effect of mutations on proper SWI/SNF function will be important for improved cancer therapy.

Paralogous subunits and their redundancy
From a therapeutic standpoint, as mutations in genes encoding SWI/SNF complex subunits are often loss-of-function, including nonsense, frameshift, and large deletions, the products of the mutant genes themselves do not constitute obvious drug targets. Consequently, it is of great interest to identify specific vulnerabilities conferred by these mutations upon cancer cells that have the potential to provide new therapeutic opportunities. One of the critical interests in this context is identification of synthetic lethality in SWI/SNF complex, owing to the many paralogous subunits it contains.

ARID1A and ARID1B are 60% identical in protein sequence and are mutually exclusive since individual SWI/SNF chromatin remodeling complexes can contain either ARID1A or ARID1B but not both. In ARID1A mutant cancer cells, ARID1B was identified as the number one dependency, suggesting that ARID1A mutation, cells become reliant upon ARID1B.  Same is the case with ATPases, cells with SMARCA4 mutation rely on SMARCA2 as the remaining SWI/SNF ATPase subunit and thus cannot tolerate loss of SMARCA2 residual complex. The mechanism by which these residual complexes contribute to tumorigenesis is not yet fully understood. One way we can begin to understand is by deciphering the exact redundant and nonredundant functions of these paralogs.
In this context as ARID1A and SMARCA4 are the most frequently mutated subunit known across many cancers we are interested to understand how different they are from their paralogs ARID1B and SMARCA2 respectively. We are also focused on the role of acetylation of SMARCA2 as we have previously found important functions of acetylation of its yeast homolog, Snf2. Yeast Snf2 targets acetylated nucleosomes through its bromodomain which binds acetyl-lysine. However, when the SAGA complex acetylates the Snf2 protein itself the bromodomain binds to acetyl-lsyines on Snf2 releasing it from the acetylated nucleosomes.
3.
Investigation of relationships between chromatin modification and metabolism.
PKM2 is a key enzyme for glycolysis and catalyzes the conversion of PEP to pyruvate, which supplies cellular energy. PKM2 phosphorylates histone H3T11. We found that the yeast PKM2 homologue, Pyk1, is part of SESAME (Serine-responsive SAM-containing Metabolic Enzyme complex). SESAME regulates the crosstalk between H3K4 methylation and H3T11 phosphorylation by sensing glycolysis and glucose-derived serine metabolism. H3Tp11 confers the resistance to oxidative stress in yeast. Furthermore, H3pT11 regulates the transcription of the genes involved in the metabolic transition to the mitochondrial respiratory pathways and the genes related to the nutrient stress responses. The CK2 and Sch9 kinases (not SESAME) are involved in signaling of H3T11 phosphorylation in response to stress and the metabolic transition toward aging. Loss of phosphorylation of H3T11, or loss of CK2 or Sch9 extends chronological lifespan. We are currently studying further molecular mechanism of chromatin modifications in metabolic regulations.
4.
Investigations of biological roles of a higher eukaryote-specific HAT complex.
We discovered the ATAC (Ada Two A Containing) HAT complex in Drosophila. ATAC is present in flies and mammals but does not exist in yeast. dATAC consists of thirteen proteins including two different HATs (Gcn5/KAT2 and Atac2/KAT14) which are conserved in humans. While Gcn5 preferentially acetylates histone H3K9 and H3K14, Atac2 is an essential H4K16 acetyltransferase. In addition, dATAC facilitates nucleosome remodeling. ATAC governs the transcriptional response to MAPK signaling by serving as a positive co-activator of transcription while also suppressing further upstream signaling.

Human MBIP is a component of human ATAC and has homology to MOCS2B (human MoaE, a molybdopterin, MPT, synthase subunit). We discovered that human ATAC and MPT synthase directly interact with protein kinase R (PKR) and suppress latent autophosphorylation of PKR, which is required for its kinase activity and its downstream phosphorylation of JNK and translation initiation factor, eIF2a. The suppression of eIF2 phosphorylation via MPT synthase and ATAC promotes translation initiation. Thus, MPT synthase and ATAC link transcription and translation initiation. MPT synthase is essential for Molybdenum (Mo) cofactor biosynthesis. We are studying the molecular linkage of Mo enzymes to chromatin regulation through the association of MPT synthase and ATAC.
5.
Investigation of mechanisms underlying onset of Alzheimer’s disease (AD) through studying Moco biosynthesis.
We have focused on finding decisive molecular linkages of metabolic disorder to onset of AD to find approaches for prevention of AD. We found that human MPT synthase functions in the complex, named MPTAC (MPT synthase Associating Complex), which promotes sulfur amino acid catabolism to prevent oxidative damage from excess sulfur amino acids. The association of MPTAC with PRMT5 arginine methytransferase complex and SNRPs splicing factors enables SNRPs to sense the metabolic states through their methylation. This promotes the splicing fidelity of APP pre-mRNA and proper APP fragmentation, abnormalities of which have been observed in the platelets of AD patients. The functions of MPTAC are crucial to maintain expression of drebrin 1, which is required for synaptic plasticity. Thus, the regulation of sulfur amino acid catabolism by MPTAC prevents events that occur early in the onset of AD. We are studying whether MPTAC play roles for onset of other neurodegenerative diseases.
6.
Functions of modules of the SAGA complex.
The SAGA complex is a 2MDa complex discovered in our lab in 1997. SAGA has histone acetylation (HAT) and histone deubiquitinase (DUB) activities. SAGA complex contains several sub-modules (e.g. HAT and DUB modules) each of which has the potential to interact with histones and chromatin through specific protein domains (e.g. Bromodomain, Tudor domains, activation interaction domain, TBP-binding domain, etc). To better understand the functions of different modules of SAGA in its recruitment and activity at target genes we have used inducible RNAi fly lines to knock down specific potential chromatin-interacting subunits in salivary glands and whole larvae. ChIP-Seq of other subunits coupled with RNA-Seq has examined the role of different modules in occupancy and activity at different loci. ChIP-Seq monitoring of H3K9 acetylation and H2B ubiquitination has ascertained effects on enzymatic activities of the complexes. Proteomic analysis of SAGA from the knock down lines monitor the integrity of the complex.

We have a particular interest in potential novel functions of the nonstop ubiquitin protease module of SAGA. Our former postdoc, Daeyoup Lee, has shown that in yeast this module is stripped from the larger SAGA complex by the 19S proteosome regulatory particle to function in mRNA export. Moreover, we have found that in ataxin-7 mutants the Drosophila ubiquitin protease module of SAGA separates from the complex yet remains active. Ataxin-7 undergoes poly Q expansion in Sca-7 disease, hence we are engineering flies carrying Ataxin-7 with expanded poly Q repeats to measure its effect on SAGA and ubiquitin protease module. We are also interested in potential roles of the ubiquitin protease module outside of SAGA. We have found and purified a subset of the ubiquitin protease module that is separate from SAGA and found novel interacting proteins. Amongst these are Arp2/3 complex components raising the possibility that the non-stop ubiquitin protease module plays a role in nuclear actin dynamics. Germline clone mutants of ataxin-7 show defects early in embryogenesis suggesting that SAGA may be important for early zygotic transcription. The key questions we are interested in here is what changes in gene expression occur in the ataxin-7 mutant and are they do to loss of SAGA function or gain of function of the dissociated nonstop ubiquitin protease module?