About

The Chatterjee lab applies chemical tools to study the mechanistic roles of key proteins found in biochemical pathways associated with human conditions, such as cancers and intellectual disability disorders. A relatively small number of proteins contribute to the vast array of biochemical pathways underlying human health and development. This requires single proteins to undertake many different functions. Protein Post-Translational Modification (PTM) by structurally and chemically unique moieties that directly change a protein’s overall shape, charge, cellular location, or catalytic activity, enable diverse functions for single proteins.

Research topics

• Biochemistry
• Chemistry
• Cell biology
• Biology

Selected publications

• Covalent hitchhikers guide proteins to the nucleus

  Cell chemical biology · 2024-03-01

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• Total chemical synthesis of sumoylated histone H4 reveals negative biochemical crosstalk with histone ubiquitylation

  Chemical Communications · 2023-01-01 · 2 citations

  articleOpen accessSenior authorCorresponding

  An efficient total chemical synthesis of site-specifically sumoylated histone H4 was undertaken to generate homogenously modified mononucleosomes. These were tested as substrates in biochemical assays with the histone H2B-specific ubiquitin ligases Rad6 and Bre1, which revealed the strong inhibition of H2B ubiquitylation by SUMO. This novel negative biochemical crosstalk between SUMO and ubiquitin was also confirmed to exist in human cells.

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• The semisynthesis of site-specifically modified histones and histone-based probes of chromatin-modifying enzymes

  Methods · 2023-05-25 · 5 citations

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• Peptide and protein chemistry approaches to study the tumor suppressor protein p53

  Organic & Biomolecular Chemistry · 2022-01-01 · 2 citations

  reviewOpen access1st authorCorresponding

  The tumor suppressor and master gene regulator protein p53 has been the subject of intense investigation for several decades due to its mutation in about half of all human cancers. However, mechanistic studies of p53 in cells are complicated by its many dynamic binding partners and heterogeneous post-translational modifications. The design of therapeutics that rescue p53 functions in cells requires a mechanistic understanding of its protein-protein interactions in specific protein complexes and identifying changes in p53 activity by diverse post-translational modifications. This review highlights the important roles that peptide and protein chemistry have played in biophysical and biochemical studies aimed at elucidating p53 regulation by several key binding partners. The design of various peptide inhibitors that rescue p53 function in cells and new opportunities in targeting p53-protein interactions are discussed. In addition, the review highlights the importance of a protein semisynthesis approach to comprehend the role of site-specific PTMs in p53 regulation.

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• Author response: Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

  2021-10-14 · 1 citations

  peer-reviewOpen accessSenior author

  Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells. Editor's evaluation This manuscript uses chemically synthesized histone H4 with K12 sumoylation coupled with in vitro transcription assay and other in vitro as well as cellular biochemical assays to provide insights about the function of histone H4 K12 sumoylation. The results suggest that H4 K12 sumoylation suppresses H4 acetylation and H3 K4 methylation, two modifications that promote transcription. The biochemistry is very nicely done and the finding may be of interest to the people interested in histone modifications. https://doi.org/10.7554/eLife.67952.sa0 Decision letter eLife's review process Introduction Chromatin is Nature’s elegant architectural solution to the challenge of packing approximately 3 billion base-pairs of human genomic DNA in an average nuclear volume of only about 500 µm3. Histones constitute the main protein component of chromatin and their reversible post-translational modifications (PTMs), or marks, regulate chromatin structure and function by a range of direct and indirect mechanisms (Kouzarides, 2007). Based upon their association with either transcriptionally active or silenced regions of chromatin, histone marks were proposed to constitute an epigenetic code for gene function (Strahl and Allis, 2000). As a consequence of their early discovery and the development of modification-specific chemical and molecular biological tools, marks such as methylation (Greer and Shi, 2012), acetylation (Shahbazian and Grunstein, 2007), and ubiquitylation (Weake and Workman, 2008) have been extensively investigated in vitro and in cell culture. In contrast, histone modification by the small ubiquitin-like modifier (SUMO) protein is a poorly understood mark due both to its very low abundance in cells, which prevents the isolation of sumoylated histones in quantities required for biochemical analysis, and to a lack of sumoylated histone-specific antibodies for cellular studies. First reported in human HEK293T and P493-6 B cells by Shiio and Eisenman, 2003, histone sumoylation also occurs in yeast (Ryu et al., 2019), parasitic protozoans (Issar et al., 2008), and plants (Miller et al., 2010). Similar to histone ubiquitylation, sumoylation occurs on all core histones, the linker histone H1, the histone variants H2A.Z and H2A.X, and the centromeric histone variant Cse4 in yeast (Hendriks and Vertegaal, 2016; Ohkuni et al., 2016). Myriad roles have been proposed for histone sumoylation in different organisms, including transcriptional regulation, kinetochore assembly, the regulation of chromatin structure, and double-strand break repair (Ryu and Hochstrasser, 2021). Pioneering efforts to identify specific lysine sites of sumoylation identified K12 in histone H4 as a major recurring site of sumoylation by SUMO-2/3 (H4K12su) (Galisson et al., 2011; Hendriks et al., 2014), although multiple proximal lysines in the H4 N-terminal tail may also be enzymatically sumoylated in vitro (Hendriks and Vertegaal, 2016). Genetic studies in yeast and human cells have typically associated H4 sumoylation with the repression of gene transcription, although mechanistic studies of the direct roles for histone sumoylation in human cells have remained intractable due to the dynamic nature and low abundance of sumoylation (Shiio and Eisenman, 2003; Nathan et al., 2006). In an effort to understand the direct effects of H4K12su in chromatin, we previously applied a disulfide-directed chemical sumoylation strategy to generate uniformly and site-specifically sumoylated nucleosome arrays (Dhall et al., 2014). Biophysical studies of chromatin-array compaction remarkably showed that H4K12su is incompatible with the compact chromatin structures seen in transcriptionally silent heterochromatin. Subsequent biochemical studies revealed that H4K12su stimulates intranucleosomal activity of the H3K4me2-specific histone demethylase LSD1 (Dhall et al., 2017). These studies suggested that sumoylated H4 does not directly enable heterochromatin formation and may instead act by recruiting LSD1 to genes. However, a potentially direct effect of histone H4 sumoylation on promoter-driven transcription by RNA polymerase II (RNAPII) and associated initiation factors that are key for efficient eukaryotic gene transcription has remained unknown. Pioneering studies of the reconstitution of class II promoter-driven accurate eukaryotic transcription in both nuclear extracts and purified systems have led to insights into roles for histone modifications in gene function (Dignam et al., 1983; Roeder, 2019). The ability to reconstitute chromatinized plasmid templates using chemically modified histones enables studies of the roles of specific histone modifications in transcription and investigations of their crosstalk with key enzymes associated with transcription initiation and elongation (Kim et al., 2009). Multiple proteins involved in gene transcription bind to and modify histone tails, which enables the remodeling of chromatin prior to and during transcription. One such modification, acetylation of lysine side-chains on H3 and H4 by the acetyltransferase p300, is necessary for efficient activator-driven transcription of both 11 nm chromatin (An et al., 2002) and 30 nm linker histone H1-containing heterochromatin, likely through mechanisms that include direct decompaction of chromatin upon H4K16 acetylation and octamer eviction by the chromatin remodeler NAP1 (Robinson et al., 2008; Shimada et al., 2019). Due to their proposed opposing roles in gene transcription, we investigated the precise nature of biochemical crosstalk between histone sumoylation and histone acetylation by p300. Histone H4 site-specifically sumoylated at Lys12 (H4K12su) was synthesized with the aid of a traceless ligation auxiliary, 2-aminooxyethanethiol (Weller et al., 2014), and then incorporated into histone octamers for subsequent reconstitution of cognate mononucleosomes and chromatinized plasmids. Each sumoylated substrate was subjected to acetyltransferase assays with the full-length p300 enzyme, which revealed the inhibition of acetylation in the H4K12su tail. Consistent with this observation and requirements for both H3 and H4 acetylation for efficient in vitro transcription of chromatin (An et al., 2002), replacing wild-type (wt) H4 with H4K12su in chromatinized plasmid templates dramatically inhibited p300-dependent, RNAPII-mediated transcription in vitro. Bottom-up mass spectrometry on chromatinized histones, following a novel in-gel desumoylation protocol, revealed decreased acetylation in H4K12su by p300 when compared to wt H4 acetylation. Consistent with a role in gene repression, H4K12su also inhibited H3K4 methylation by the extended catalytic module (eCM) of the Set1/COMPASS (complex of proteins associated with Set1) methyltransferase complex (Hsu et al., 2019). To confirm the negative crosstalk with SUMO in human cells, linear non-hydrolyzable genetic fusions of SUMO-H4 were generated in HEK293T cells and analyzed by western blotting and chromatin immunoprecipitation followed by high-throughput sequencing of the associated DNA (chromatin immunoprecipitation [ChIP]-seq). Collectively, our observations provide the first unambiguous biochemical demonstration that sumoylated histone H4 directly inhibits RNAPII-mediated transcription from chromatin templates, and reveal its direct negative crosstalk with histone acetylation by p300 and methylation by Set1/COMPASS that are strongly associated with active gene transcription. Results Reconstitution of site-specifically sumoylated octamers and nucleosomes Site-specifically sumoylated human histone H4 at Lys12, H4K12su, was obtained by a semisynthetic strategy using the ligation auxiliary 2-aminooxyethanethiol (Figure 1A and B; Dhall et al., 2017). The semisynthetic sumoylated H4 was incorporated into octamers with purified recombinant human histones H2A, H2B, and H3 (Figure 1C and Figure 1—source data 1). Nucleosomes were reconstituted from sumoylated octamers using the 147 bp Widom 601 double-stranded DNA (dsDNA) (Figure 1D). Figure 1 with 1 supplement see all Download asset Open asset Sumoylation inhibits p300-mediated H4 acetylation in octamer and mononucleosome substrates. (A) Synthetic scheme for H4K12su. (i) An H4(1–14)K12aux peptide was ligated with a SUMO-3 (2–91) C47S α-thioester. (ii) The sumoylated H4(1–14) peptidyl hydrazide containing the auxiliary was converted to a C-terminal α-thioester and ligated with H4(15–102) A15C. The auxiliary was then reductively cleaved from the ligation product. Cys15 in the final ligation product was desulfurized to the native Ala15 to yield site-specifically sumoylated H4K12su. (B) C4 analytical RP-HPLC trace of purified H4K12su (top). ESI-MS of purified H4K12su (bottom). Calculated mass 21,596.7 Da. Observed, 21,594.2 ± 3.4 Da. (C) Coomassie-stained 15% SDS-PAGE of reconstituted octamers containing wild-type (wt) H4 or H4K12su. (D) Ethidium bromide stained 5% TBE gel of mononucleosomes containing wt H4 or H4K12su. (E) Western blots of p300 assay products with octamer substrates containing wt H4 or H4K12su, probed with a site-independent pan-acetyllysine antibody (top) and an H4K16ac-specific antibody (bottom). An asterisk indicates assays with heat-inactivated p300 to exclude non-enzymatic acetylation. (F) Fluorogram of p300 assay products with [3H]-acetyl-CoA as the co-factor and mononucleosome substrates containing wt H4 or H4K12su. Figure 1—source data 1 Unedited intact SDS-PAGE gels for all gel images shown in Figure 1. https://cdn.elifesciences.org/articles/67952/elife-67952-fig1-data1-v2.pdf Download elife-67952-fig1-data1-v2.pdf Histone octamer acetylation by p300 We previously showed that H4K12su stimulates activity of the H3K4me1/2 demethylase, LSD1, in the context of an LSD1-CoREST sub-complex (Dhall et al., 2017). The stimulation of histone deacetylase (HDAC) activity of the Set3c complex in yeast was also recently proposed for sumoylated histone H2B (Ryu et al., 2020). Although the erasure of specific methyl and acetyl marks in the H3 and H4 tails may facilitate the transcriptionally repressed state of chromatin, there remains no information regarding the re-installation of these marks by the corresponding writer enzymes in the presence of H4K12su. Key among the histone acetyltransferases (HAT) is the enzyme p300 that is recruited to chromatin by transcriptional activators and undertakes histone tail acetylation prior to transcription initiation (An et al., 2002; Kraus and Kadonaga, 1998; Kundu et al., 2000). Given its essential role in transcription, we investigated the effect of H4K12su on histone acetylation by p300 prior to and during transcription by RNAPII. To investigate the direct biochemical crosstalk between H4K12su and acetylation, a western blot-based HAT assay was developed using a sequence-independent pan-acetyllysine antibody to detect lysine acetylation in all four histones (Figure 1—figure supplement 1A and Figure 1—figure supplement 1—source data 1). In order to effectively compare acetylation of wt H4 with H4K12su and to strictly exclude any acetylation of the surface-exposed lysines in SUMO-3 attached to H4 (Figure 1—figure supplement 1B), it was proteolyzed from H4K12su prior to western blot analysis. To this end, after acetylation by p300, the assay product was heat-inactivated at 65°C for 10 min, followed by addition of the purified catalytic domain of human SENP2 containing residues 365–590 (Figure 1—figure supplement 1C; Mikolajczyk et al., 2007). Heat inactivation precluded p300 activity during desumoylation, and enabled the direct comparison of acetylation status in H4 and H4K12su. Histone octamers containing wt H4 were first acetylated with full-length p300 immuno-affinity purified from HEK293T cells with an N-terminal FLAG epitope-tag (Figure 1—figure supplement 1D; Chen et al., 2002). Western blot analysis showed the robust acetylation of all four histones (Figure 1E top panel and Figure 1—figure supplement 1E). This is consistent with previous in vitro assays that revealed acetylation of all four histones by p300 (Ogryzko et al., 1996). Strikingly, H4 from octamers containing H4K12su was devoid of acetylation, including H4K16ac (Figure 1E bottom panel), which is strongly associated with chromatin decompaction and active gene transcription (Robinson et al., 2008; Akhtar and Becker, 2000; Shogren-Knaak et al., 2006). Importantly, the inhibition of H4 tail acetylation does not arise from allosteric inactivation of p300, deduced from our observation that H2A/H2B/H3 in H4K12su octamers were acetylated to the same extent as in wt H4 octamers. Additionally, western blots confirmed that SUMO-3 did not inhibit p300 autoacetylation, which is associated with robust acetyltransferase activity (Figure 1—figure supplement 1F). Hence the inhibition of H4 acetylation in H4K12su is likely due to lysine acetylation site occlusion by proximal SUMO-3 in the H4 tail. Mononucleosome acetylation by p300 The histone acetylation assay was next undertaken with mononucleosomes containing either wt H4 or H4K12su. We failed to see significant nucleosome acetylation with pan-acetyllysine antibodies with or without pre-incubation of p300 with acetyl-CoA prior to the addition of nucleosomes (Figure 1—figure supplement 1G). Due to the significantly decreased activity of p300 with nucleosomal substrates, a [3H]-acetyl-CoA co-factor was employed and the transfer of acetyl groups to histones observed by fluorography. H4 acetylation was also suppressed in mononucleosomes containing H4K12su, but not in unmodified H4 mononucleosomes (Figure 1F). Our results unequivocally suggested that SUMO-3 in the H4 tail is inhibitory toward p300-mediated acetylation of chromatin, a process that is necessary for active gene transcription. Based on the lower acetyltransferase activity observed with mononucleosomes than with octamers, we wondered if dsDNA may inhibit p300 activity. To test this, an equimolar amount of free 147 bp Widom 601 dsDNA was included in the octamer acetylation assay. The presence of DNA was sufficient to inhibit p300 activity to a similar extent as observed with mononucleosomes (Figure 1—figure supplement 1H). This unexpected inhibition of p300 activity by free DNA suggests that additional factors, such as transcription factor and RNAPII binding, enable robust p300 activity on histones during transcription initiation and elongation. The effect of H4K12su on cell-free transcription from chromatinized templates Based on our observation that H4K12su inhibits the acetylation of key H4 tail residues that are associated with p300-dependent active transcription, including H4K16 that is acetylated in euchromatin, we sought to investigate the direct effect of H4K12su on transcription in our reconstituted cell-free system. These assays employed a p300/Gal4-VP16-dependent transcription system using chromatinized plasmids assembled with reconstituted octamers containing either wt H4 or H4K12su (Figure 2A). The plasmid DNA template consisted of five gal4 binding sites and an ~400 bp G-less cassette (Kundu et al., 2000). Due to the absence of any engineered strong nucleosome positioning sequences in the template, chromatinization was undertaken with the histone chaperone NAP1 and the chromatin remodelers Acf1 and ISWI (Figure 2—figure supplement 1 and Figure 2—figure supplement 1—source data 1). Limited micrococcal nuclease (MNase) digestion of the transcription templates revealed the periodic spacing of nucleosomes in chromatin assembled with either wt H4 or H4K12su octamers, and clearly indicated that H4K12su does not inhibit the formation of recombinant chromatin (Figure 2B and Figure 2—source data 1). In this background, addition of the transcription activator Gal4-VP16, p300, acetyl-CoA, [α-32P]-CTP, rNTPs, and transcriptional machinery from a HeLa nuclear extract resulted in the transcription of a 365 base RNA from the chromatin template assembled with wt H4 histones (Figure 2C). Surprisingly, and in contrast to the direct structural decompaction of chromatin by H4K12su, transcription from templates assembled with H4K12su was drastically inhibited when compared with templates assembled with wt H4. The addition of trichostatin A, a nanomolar inhibitor of class I and II HDACs, did not lead to significant changes in transcription, indicating that the repressive effect of H4K12su is not significantly mediated through HDAC1 in chromatinized templates assembled with non-acetylated histones (Schultz et al., 2004). Importantly, our results unambiguously demonstrated transcriptional repression when site-specifically sumoylated H4 was present in chromatin. Figure 2 with 1 supplement see all Download asset Open asset Histone H4 sumoylation inhibits in vitro transcription from chromatinized plasmid templates. (A) Scheme outlining steps during the in vitro transcription assay with chromatinized plasmids, nuclear extracts, activator Gal4-VP16 and p300. (B) Micrococcal nuclease digestion analysis of plasmids chromatinized with wild-type (wt) H4 or H4K12su indicating the similar occupancy and spacing of mononucleosomes. (C) Autoradiogram of 32P-labeled 365 base RNA transcript generated from p300-mediated transcription from chromatinized templates containing wt H4 or H4K12su in the presence or absence of the histone deacetylase (HDAC) inhibitor, trichostatin A. Figure 2—source data 1 Unedited intact TBE gel for chromatin digestion gel shown in Figure 2. https://cdn.elifesciences.org/articles/67952/elife-67952-fig2-data1-v2.pdf Download elife-67952-fig2-data1-v2.pdf H4 acetylation is inhibited prior to gene transcription in chromatin containing H4K12su Based on our observations with sumoylated octamer and nucleosome substrates, we wondered if the inhibition of transcription by H4K12su also correlated with diminished H4 tail acetylation by p300. Previous in vitro transcription studies with chromatinized plasmids containing either K-to-R mutations in the H4 tail or truncated H4 missing tail residues 1–19 revealed an ~80% reduced transcriptional output relative to transcription from chromatin containing wt H4 (An et al., 2002). Chromatinized plasmids containing either wt H4 or H4K12su were incubated with Gal4-VP16, p300, and acetyl-CoA for 30 min to enable steps preceding transcription; and the histones were subsequently resolved by SDS-PAGE and analyzed by tandem mass spectrometry (MS-MS) after chemical propionylation, trypsination, and separation by capillary-liquid chromatography (Figure 3—figure supplement 1; Sidoli and Garcia, 2017). A critical innovation in our bottom-up analysis workflow was the use of a SENP2 catalytic domain to desumoylate H4K12su within the polyacrylamide gel matrix after SDS-PAGE. This procedural step was required to generate the same H4(4–17) tryptic peptide from wt H4 and H4K12su after p300-mediated acetylation. The H4(4–17) peptide contains K5,8,12, and 16 that are known to be acetylated by p300 in vitro and in vivo (Ogryzko et al., 1996). Analysis of the H4(4–17) tryptic peptides arising from wt H4 revealed a remarkable degree of hyperacetylation within 30 min. The most abundant peptide corresponded to the K5,8,12,16 tetra-acetylated form with some tri-acetylated species also present (Figure 3—source data 1, Figure 3A, Table 1, and Figure 3—figure supplements 2 and 3). This is consistent with the fact that p300 acetylates histones to facilitate transcription (An et al., 2002; Robinson et al., 2008; Shimada et al., 2019; Kundu et al., 2000). No significant degree of monoacetylation was observed, and a low abundance of diacetylated peptide was detected after manually searching the MS-MS spectra over the expected elution time-range (Figure 3—figure supplement 4). In comparison, chromatin assembled with H4K12su generated significantly fewer hyperacetylated peptides, with approximately equal amounts of tri- and di-acetylated H4(4–17) peptides (Figure 3B and Figure 3—figure supplements 5 and 6). Small amounts of unmodified H4(4–17) peptides were also observed (Figure 3—figure supplement 7). This clearly indicates that H4K12su directly inhibits p300-mediated H4 tail acetylation in the steps prior to transcription. Given the importance of H4 tail acetylation for efficient transcription, H4K12su may inhibit transcription, in part, by directly inhibiting p300 activity on H4. Table 1 H4(4–17) tail peptides acetylated by p300 in chromatinized plasmid templates with activator Gal4-VP16*,†. H4(4–17) peptide[M][M + 2 H]2+PSMH4PSMH4K12suprGKprGGKprGLGKprGGAKprR1549.89775.95n.d.1prGKprGGKacGLGKprGGAKprR1535.88768.95n.d.n.d.prGKacGGKacGLGKprGGAKprR1521.86761.9423prGKacGGKacGLGKacGGAKprR1507.85754.935n.d.‡prGKacGGKacGLGKprGGAKacR1507.85754.93n.d.3§prGKacGGKacGLGKacGGAKacR1493.83747.927n.d.‡ * Peptides were chemically propionylated before and after trypsinization to cap unmodified lysine side-chains and newly generated N-termini. † Tandem mass spectrometry (MS-MS) spectra observed contained major fragments for the shown modification pattern over other potential patterns, however, no singly acetylated peptides were observed for wild-type (wt) H4. ‡ Acetylation at K12 is not possible for H4K12su. § The triply acetylated peptide from H4K12su is blocked from acetylation at K12, but is propionylated after in-gel desumoylation. PSM = peptide spectral match. n.d. = not detected. Figure 3 with 7 supplements see all Download asset Open asset Comparison of H4 tail acetylation by p300 in chromatinized plasmid templates with activator Gal4-VP16. (A) Extracted ion chromatograms of all H4(4–17) tryptic peptides obtained after SDS-PAGE resolution and in-gel trypsination of acetylated chromatin containing wild-type (wt) H4. (B) Extracted ion chromatograms of all H4(4–17) tryptic peptides obtained after SDS-PAGE resolution, in-gel desumoylation and trypsination of acetylated chromatin containing H4K12su. The extracted m/z of each spectrum is centered on the [M + 2 H]2+ precursor ion. Figure 3—source data 1 Excel file listing all mass spectral data plotted in Figure 3, Tables 1–4; and Figure 3—figure supplements 2–7. Download all four acetylated of H4(4–17) were observed during analysis of p300 assay products with and chromatin substrates, we the site of p300 in the H4 tail. Consistent with previous we observed that and are sites in the acetylated H4 over acetylation at K12 and et al., Additionally, K12 was acetylated over in the triply acetylated H4 tail peptide 3). These observations were consistent between H4 or H4K12su containing substrates, indicating that the substrate of p300 is in the presence of SUMO-3 4). Table 2 of relative ion of from an enzymatically di-acetylated and chemically propionylated H4(4–17) [M + 2 H]2+ = after activator and p300-mediated acetylation of chromatinized plasmids containing wild-type (wt) of ion * two spectra were observed and analyzed for the acetylated and propionylated H4(4–17) tail peptide from wild-type (wt) H4 chromatin. Table 3 of relative ion of from an enzymatically tri-acetylated and chemically propionylated H4(4–17) [M + 2 H]2+ = after activator and p300-mediated acetylation of chromatinized plasmids containing wild-type (wt) of ion ± ± * spectra corresponding to the tri-acetylated and propionylated H4(4–17) tail peptide from wild-type (wt) H4 chromatin were reported is of the n.d. = not detected. Table of relative ion of from an enzymatically and chemically propionylated H4(4–17) [M + 2 H]2+ = after activator and p300-mediated acetylation of chromatinized plasmids containing of ion ± * spectra corresponding to the di-acetylated and propionylated H4(4–17) tail peptide from H4K12su chromatin were reported is of the n.d. = not detected. Biochemical crosstalk between H4 sumoylation and acetylation in human cells In order to the biochemical between histone H4 sumoylation and acetylation in human cells, HEK293T cells were with a plasmid an gene This fusion SUMO-3 at the of H4 proximal to K12 in the tail also intact for antibodies or To desumoylation by the two C-terminal residues in SUMO-3 were to generate the fusion protein of nuclear chromatin was and with to mononucleosomes (Figure nucleosomes containing either or two of were using and the acetylation state of and H4K16 was investigated in western blots using antibodies (Figure data 1). This analysis revealed that the of SUMO-3 to the of H4 significantly inhibits H4K16ac and has a inhibitory effect on (Figure the K12 were then K12 also be blocked from acetylation. The presence of all core histones in the also demonstrated that the fusion was incorporated into chromatin within human cells (Figure supplement 1). Thus, the biochemical observed in HEK293T cells is consistent with our observation that H4K12su inhibits H4K16ac in and indicates a degree of in the precise site of sumoylation that may negative biochemical crosstalk with These cellular studies also our discovery that H4 tail sumoylation inhibits p300 activity to include other known H4K16 acetyltransferases such as et al., and and 1996). Figure with 1 supplement see all Download asset Open asset Biochemical crosstalk between H4 sumoylation and acetylation in HEK293T cells. (A) micrococcal nuclease digestion of chromatin to generate mononucleosomes that were detected by the presence of bp DNA in (B) from cells with and (E) to chromatin and mononucleosomes containing and H4K16ac were employed to detect the degree of wild-type (wt) H4 and acetylation in mononucleosomes. histone H3 in each was employed as an

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• Histone Synthesis

  2021-02-16 · 1 citations

  other1st authorCorresponding

  Histones are the major protein component of eukaryotic chromatin that not only act as packaging proteins for nuclear DNA but also participate in biochemical and biophysical processes that regulate gene function, replication, and repair. Our understanding of the many mechanistic roles for histones has rapidly expanded in the past two decades, due in large part to the addition of semisynthetic and fully synthetic site-specifically modified histones to the repertoire of tools available to investigate the histone code hypothesis. This chapter will introduce readers to the wide range and functional diversity of histone modifications. Cellular and organismal studies that have indicated a biochemical relationship, or crosstalk, between sets of modifications will be presented. In this context, the advantages of native chemical ligation and the closely related expressed protein ligation will be discussed. Finally, several biochemical and biophysical studies will be presented with a focus on the key advancements in histone synthesis that have enabled facile access to milligram quantities of homogenous histone reagents.

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• BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1

  Nature Structural & Molecular Biology · 2021 · 87 citations

  • Cell biology
  • Biology
  • Chemistry
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• Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

  bioRxiv (Cold Spring Harbor Laboratory) · 2021-04-08 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Abstract Post-translational modification of histone H4 by the small ubiquitin-like modifier (SUMO) protein was associated with gene repression. However, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using H4 chemically modified at Lys12 by SUMO-3 (H4K12su) that was incorporated into mononucleosomes and chromatinized plasmids. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, and transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed H4K12su inhibits RNA polymerase II-mediated transcription and H4 tail acetylation. Thus, we have uncovered negative biochemical crosstalk with acetylation/methylation and the direct inhibition of RNAPII-mediated transcription by H4K12su.

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• Tackling a Curious Case: Generation of Charge-Tagged Guanosine Radicals by Gas-Phase Electron Transfer and Their Characterization by UV–vis Photodissociation Action Spectroscopy and Theory

  Journal of the American Society for Mass Spectrometry · 2021-02-10 · 9 citations

  articleOpen access

  We report the generation of gas-phase riboguanosine radicals that were tagged at ribose with a fixed-charge 6-(trimethylammonium)hexane-1-aminocarbonyl group. The radical generation relied on electron transfer from fluoranthene anion to noncovalent dibenzocrown-ether dication complexes which formed nucleoside cation radicals upon one-electron reduction and crown-ether ligand loss. The cation radicals were characterized by collision-induced dissociation (CID), photodissociation (UVPD), and UV–vis action spectroscopy. Identification of charge-tagged guanosine radicals was challenging because of spontaneous dissociations by loss of a hydrogen atom and guanine that occurred upon storing the ions in the ion trap without further excitation. The loss of H proceeded from an exchangeable position on N-7 in guanine that was established by deuterium labeling and was the lowest energy dissociation of the guanosine radicals according to transition-state energy calculations. Rate constant measurements revealed an inverse isotope effect on the loss of either hydrogen or deuterium with rate constants kH = 0.25–0.26 s–1 and kD = 0.39–0.54 s–1. We used time-dependent density functional theory calculations, including thermal vibronic effects, to predict the absorption spectra of several protomeric radical isomers. The calculated spectra of low-energy N-7-H guanine-radical tautomers closely matched the action spectra. Transition-state-theory calculations of the rate constants for the loss of H-7 and guanine agreed with the experimental rate constants for a narrow range of ion effective temperatures. Our calculations suggest that the observed inverse isotope effect does not arise from the isotope-dependent differences in the transition-state energies. Instead, it may be caused by the dynamics of post-transition-state complexes preceding the product separation.

  PublisherOA PDFDOI

• Development of Tyrphostin Analogues to Study Inhibition of the <i>Mycobacterium tuberculosis</i> Pup Proteasome System**

  ChemBioChem · 2021-08-12 · 13 citations

  articleOpen access

  Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.

  PublisherOA PDFDOI

Recent grants

• Chemical Strategies to Investigate Gene Regulation By Histone Sumoylation

  NIH · $306k · 2014–2023

• Structure and Mechanism of the SET1/COMPASS H3K4 Methyltransferase Complex

  NIH · $1.9M · 2020–2025

• Chemical Strategies to Investigate Gene Regulation By Histone Sumoylation

  NIH · $2.2M · 2014–2024

• Testing the p53 code for Gene Regulation with Chemical Protein Synthesis

  NSF · $450k · 2021–2025

• Chemical strategies to investigate biochemical crosstalk in human chromatin

  NIH · $1.6M · 2023–2028

Frequent coauthors

• Caroline E. Weller

  15 shared

• Ning Zheng

  University of Washington

  14 shared

• Peter Hsu

  11 shared

• Abhinav Dhall

  11 shared

• Samuel D. Whedon

  Brigham and Women's Hospital

  10 shared

• Robert K. McGinty

  10 shared

• Tom W. Muir

  Princeton University

  10 shared

• Wilfred A. van der Donk

  Howard Hughes Medical Institute

  10 shared

Labs

• Chatterjee Research LabPI

  Not provided

Education

• Ph.D., Chemistry

  University of Illinois at Urbana-Champaign

  2005

Awards & honors

• American Peptide Society Fellow
• European Peptide Society Fellow
• Champak Chatterjee promoted to Associate Professor with Tenu…