Siddhartha Roy Group
Laboratory of Protein Biochemistry, Biophysics and Structural Biology
at CSIR - Indian Institute of Chemical Biology
We seek to understand the role of certain macromolecules in the regulation of transcription and chromatin dynamics which crucially impact many fundamental biological processes including development and disease. The ongoing research of the laboratory is focused on dissecting the molecular basis of chromatin organization by means of mediating histone modifications by writer enzymes and histone assembly by histone chaperones - with a combined approach of structural biology, biochemistry, and biophysical approaches.
Elucidating the mechanism of action of epigenetic 'writer' enzymes and their role in regulation of chromatin dynamics and diseases
Within the eukaryotic nucleus, chromatin exists as a dynamic structure which is subjected to constant reversible modifications by epigenetic regulators for efficient DNA mediated mechanisms. Apart from DNA methylation, histone modifications play crucial role in altering DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. Chromatin, which is formed of functional units called nucleosomes, are composed of four core histones H2A, H2B, H3 and H4, wrapped around by 147 base pairs of DNA. While the globular histone cores are packed within the nucleosome, the N-terminal (and in some cases, the C-terminal) histone tails protrude from the nucleosome and are subjected to a variety of posttranslational modifications (PTMs). PTMs can also target the exposed surface of the histone cores that are in contact with the DNA. These modifications modulate the chromatin structure by altering the net charge, inter-nucleosomal interactions and by assisting the recruitment of specific complexes involved in chromatin remodeling and are catalyzed by enzymes known as 'writers' of such epigenetic marks. Of the varied PTMs that had been reported, we are currently interested in histone methylation and ubiquitination.
From previous studies, it is already known that histones, predominantly H3 and H4, are methylated at a number of lysine (Lys) and arginine (Arg) residues. Methylation can occur in the form of mono, di, or trimethylation, and this differential methylation assigns particular functional significance for each and every type. Protein arginine methylation is mediated by a family of enzymes called Protein Arginine Methyltransferases (PRMTs) and catalyze asymmetric di-methylation (Type I), symmetric di-methylation (Type II) and mono-methylation only (Type III) respectively, on arginine residues in histone and non-histone proteins.
Histone arginine methylations are reported to be involved in transcriptional regulation, RNA processing and signal transduction. PRMTs have been found to be associated with various human diseases, such as cancer and inflammation. However, in spite of being an attractive potential target for anti-cancer therapy, there are no reports of suitable inhibitors against some of the PRMT family members which led us to take up structure based designing of inhibitor molecules against those members, and testing their catalytic activity in both in vitro as well as in in vivo models. By gaining structural insights into the PRMT-inhibitor complex, we plan to find out details about the binding mode of the protein and ligand for biochemical and cellular potency improvement.
Aside from methylation, we are also interested histone ubiquitination, which stands out from the other PTMs due to their enormous size and complexity. Within the nucleosome, histones H2A and H2B are commonly mono-ubiquitinated, which causes alteration in nucleosomal dynamics and initiates crosstalk with other histone modifications such as H3K4 and H3K79 trimethylation. Monoubiquitination of histone H2B at Lysine 120 (K120) in human is previously reported to be mediated by RNF20/40 complex and is associated with transcriptional activation, elongation and memory. Our lab was part of a collaboration with Dr. Chandrima Das of Saha Institute of Nuclear Physics, Kolkata and Dr. Kunal Rai of MD Anderson Cancer Center, Texas, that reported in 2019 for the first time that Ubiquitin protein ligase E3 component N-Recognin 7 (UBR7) was a novel E3 ligase which monoubiquitinates H2B at Lys120 position and acts as a tumor metastasis suppressor in triple negative breast cancer (TNBC) model. UBR7 is the smallest member of the UBR protein family of mammalian E3 ligases, which is so named due to the presence of the common zinc finger UBR box in each of the members. UBR7 has negligible size or sequence similarity with the other members and harbors a Plant Homeodomain (PHD) exclusively, which is a known chromatin binding module commonly reported to be a reader of unmodified, acetylated and methylated histones. This study provided an initial insight into the previously unknown role of PHD fingers in catalysis of histone PTMs. However, TNBC is one of the most aggressive form of breast cancer with a poor prognosis than other forms of cancers and currently has no available targeted therapy, which emphasizes the dire need to understand the mechanism of UBR7 function and its association with histones in chromatin context. Hence, we are currently interested in characterizing the association of UBR7 with its E2 partner and its substrate, and their roles in the monoubiquitination process. We are also looking into how the catalytic mechanism of UBR7 differs from the other E3 ligases involved in H2B monoubiquitination. Molecular insights into UBR7 mediated Ubiquitin transfer mechanism would shed light on its tumor metastasis suppressor potential as well.
Adhikary S, Chakravarti D, Terranova C, Sengupta I, Maitituoheti M, Dasgupta A, Srivastava DK, Ma J, Raman AT, Tarco E, Sahin AA, Bassett R, Yang F, Tapia C, Siddhartha Roy, Rai K, Das C. Atypical plant homeodomain of UBR7 functions as an H2BK120Ub ligase and breast tumor suppressor. Nat Commun. 2019 Mar 28;10(1):1398.
https://doi.org/10.1038/s41467-019-08986-5
Dasgupta A, Mondal P, Dalui S, Das C and Siddhartha Roy. Molecular characterization of substrate-induced ubiquitin transfer by UBR7-PHD finger, a newly identified histone H2BK120 ubiquitin ligase. FEBS J. 2022 Apr;289(7):1842-1857.
Dissecting the different modes of H3-H4 binding by histone chaperones for histone deposition and nucleosome assembly
Nucleosomes in their compact state restrict DNA accessibility, all the while remaining highly dynamic with respect to their positioning and state of assembly to allow access to the base readout of DNA. Several key DNA mediated processes like replication, transcription and repair all involve step wise chromatin disruption and restoration, facilitated by the histone chaperone network. Histone chaperones are proteins which bind with histones and regulate nucleosome assembly. Histone chaperones can be classified as H3–H4 or H2A–H2B chaperones, or both - based on their preferential binding. They also provide specificity by distinguishing between canonical histones and variants according to need for incorporation of nascent histones and histone pool maintenance throughout the cell cycle and in specialized chromosome domains. The histone chaperones adopt similar structural folds and share sequence homology with their preferential canonical histones.
The first histone chaperone to be crystallized was Anti-silencing function 1 (Asf1) with H3–H4, which led to the finding that Asf1 binds H3–H4 dimers through the H3 α2–α3 helices which constitute the H3–H4 tetramerization interface, thus preventing tetramer assembly. After synthesis in the cytosol, Asf1 - among other chaperones, binds to the newly formed H3–H4 dimers which are then imported from the cytoplasm to the nucleus. Various studies have shown that one molecule of Asf1 binds a H3–H4 heterodimer to form a heterotrimeric complex. Asf1 is an important upstream H3-H4 specific chaperone implicated in both the replication-coupled and independent H3-H4 assembly and thus interacts with two different chaperones , CAF1 (which deposits H3.1-H4 onto DNA in a replication dependent manner) and HIRA or DAXX (which deposits H3.3-H4 onto DNA in a replication independent manner). Interestingly, Asf1 interacts with HIRA and CAF1 with mutually exclusive surfaces. Although Asf1 from yeast and human has been structurally well characterized previously, ASF1 from the unicellular pathogenic microorganisms remains largely less studied.
Malaria is a mosquito-borne infectious disease which is caused by a unicellular protozoa of the Plasmodium genus. Among the different species, Plasmodium falciparum is reported to cause most deaths world-wide. Recent studies have shown that the malarial parasite has started developing considerable resistance to the already available anti-malarial drugs. The genome of Plasmodium falciparum is unique among eukaryotes due to high AT content and its unique proteome signature causes many of its proteins to be structurally divergent which makes its adaptive survival and propagation strategies possible in both human and mosquito hosts. We are interested to study the regulation of chromatin assembly in Plasmodium falciparum in search for potential anti-malarial drug targets.
For this, we chose PfAsf1 as the focal point of this study since it is implicated in DNA replication and repair, transcriptional regulation and chromatin silencing in higher eukaryotes. In a recent study from our lab, we have structurally and biochemically characterized PfAsf1 and found it to interact specifically with histone H3 and H4 from Plasmodium falciparum and is able to deposit H3-H4 onto DNA template to form disomes thereby acting as a histone chaperone. PfAsf1 is expressed in all the stages of the Plasmodium life-cycle. The structure of PfAsf1 that we obtained by X-Ray diffraction method indicated that it has a conserved beta-sandwich fold and may exist as a dimer. To summarize, our study has shed light on the previously unknown mechanism of Asf1 mediated chromatin assembly and disassembly process in the the uniquely evolved malarial parasite Palsmodium falciparum.
Srivastava DK, Gunjan S, Das C, Seshadri V, Siddhartha Roy. Structural insights into histone chaperone Asf1 and its characterization from Plasmodium falciparum. Biochem J. 2021 Mar 12;478(5):1117-113
We are also interested in NAP superfamily histone chaperones which have been reported to be involved in shielding functional histone interfaces, thereby shuttling both core and linker histones from their site of synthesis in the cytoplasm to the nucleus . NAP1-like structural fold is among the few signature structural folds are recognized specifically for their histone chaperone function. It forms a constitutive homodimer with a headphone-like arrangement which binds histone H3-H4 tails. This has been well characterized in 2 members of NAP Superfamily in Yeast - Vps75 and Nap1 with respect to their binding to H3–H4 and for binding of Nap1 to H2A–H2B — which is exclusive to the H3–H4 tetramerization interface. Hence, Vps75 and Nap1 can either bind H3–H4 tetramers or H3–H4 dimers in complex with Asf1. Both chaperones are capable of binding both H2A–H2B and H3–H4 in vitro. However, the binding preference of NAP domains to histones and their contextual stoichiometry is still not well understood.
In humans and higher mammals, Testis-specific protein Y-encoded-like (TSPYL) family proteins are a group of X-linked genes encoding for nuclear proteins of the TSPY-L nucleosome assembly protein-1 superfamily, which harbors a common NAP domain for putative nucleosome remodeling and gene expression regulation function. They are also implicated in Sudden Infant Death with Dysgenesis of the Testes syndrome (SIDDT), a condition in infant males that causes death in the form of cardiac and respiratory arrest, apart from testis dysgenesis, and is associated with TSPYL loss of function. However, nothing much is known about their role in regulating gene expression. Due to the involvement of TSPYL family members in various cancers and developmental disorders, we intend to study the association of their NAP domains with chromatin and their histone chaperone activity and dissect their role in modulating chromatin dynamics.
Dalui S, Dasgupta A, Adhikari S, Das C and Siddhartha Roy. Human testis-specific Y-encoded like protein 5 is a histone H3/H4 specific chaperone which facilitates histone deposition in vitro.J. Biol Chem. 2022 Aug;298(8):102200.