Ildiko Felletar - Profile on Academia.edu (original) (raw)
Papers by Ildiko Felletar
Abstract 1690: A histology based approach to spatial single cell analysis of colorectal cancers
Cancer Research, Jun 15, 2022
Background: Single cell sequencing is a powerful tool for the evaluation of intratumoral heteroge... more Background: Single cell sequencing is a powerful tool for the evaluation of intratumoral heterogeneity and the investigation of cancer evolution. Aims: By combining laser microdissection and single cell sequencing, we aimed to link tissue morphology and spatial information with sequencing data of the isolated cells. Materials & methods: In our preliminary study, we used fresh frozen tissue specimen of surgically resected material from a colorectal cancer (CRC) patient containing both cancerous and normal adjacent tissue (NAT). From part of the normal and cancerous tissue exome sequencing was performed in bulk (to assess somatic variants), while the other part was subjected to single cell sequencing. Fresh frozen tissues from both CRC and NAT were cryosectioned at -20°C with section thickness ranging from 16 to 25 µm to ensure that a layer of whole cells are present in the slides. Tissue slides were then scanned using a PANNORAMIC 1000 scanner (3DHISTECH Ltd.). After morphologic evaluation single normal colonocytes and cancerous cells were laser microdissected from the NAT and multiple CRC areas (invasive front, differentiated, non-differentiated cells) by using a CellCut Laser Microdissection system (MMI). The isolated cells were subjected to Repli-G Single Cell WGA Kit (Qiagen) and library preparation followed by whole exome sequencing (WES) on NextSeq 550 (Illumina). Blood sample was also collected before surgical treatment, cell-free DNA was isolated and exome sequencing was completed. Bioinformatic analysis was conducted using BaseSpace and GATK4 best practices. Common and unique variants were identified between cells and also compared with the bulk exom and cell free DNA sequencing results of the patient. Identified variants were further investigated using the oncoKB annotator. Results: Both healthy (1) and cancerous epithelial cells (3) were dissected and sequenced successfully. A median depth of coverage of 192 was achieved with a median of 43.3% of coverage of 50x or above in the target region compared to 73.9 and 52.8% 208 and 97.6% in tissue and plasma samples, respectively. Overall, we identified 105, likely oncogenic” and 2, predicted oncogenic” unique variants in the single cells using the oncoKB annotator. Among the cancerous single cells, we identified 10, likely oncogenic” and 1, predicted oncogenic” common variants (such as ARID4B, DNMT3B, MSH6 and HNF1A), while 15, 25 and 7, likely oncogenic” (such as CTLA4, MLH1, MSH2, CDK12, CDKN1B) variants were identified uniquely in the 3 cancerous cells. Conclusion: We were able to dissect, isolate and sequence single cells from CRC and NAT thus combining valuable morphologic information with sequencing data on a single cellular level with maintained spatial information. The distribution of variants among the single cells shows that it is a viable approach to investigate tumor heterogeneity and to link the morphologic phenotypes and genotypes of cancerous cells. Citation Format: William Jayasekara Kothalawala, Alexandra Kalmár, Gitta Szabó, Barbara Kinga Barták, Sára Zsigrai, Zsófia Brigitta Nagy, Ildikó Felletár, Krisztina Andrea Szigeti, István Takács, Béla Molnár. A histology based approach to spatial single cell analysis of colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1690.
Abstract 3745: Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression
Cancer Research, Jun 15, 2022
Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer t... more Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer types, including colorectal cancer. Abnormality of several factors, such as DNA methyltransferases (DNMT), demethylases, or deviation in methyl-donor (folate and S-adenosylmethionine) availability can contribute to the development of genome-wide hypomethylation. Detection of epigenetic changes as global DNA hypomethylation in cell-free DNA fraction obtained from blood samples can expand the opportunities for the early recognition of colorectal cancer. One of our main goals was the investigation of global DNA methylation patterns in tissue biopsies (n=183) and cell-free DNA fraction of blood samples (n=48) along the colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore possible underlying mechanisms of genome-wide hypomethylation formation in 12 colorectal tumor tissue sections, containing transition zones. Using LINE-1 pyrosequencing, significantly reduced global DNA methylation level was detected in line with cancer progression in tissue specimens (normal: 77.5±1.7%, adenoma: 72.7±4.8%, carcinoma: 69.7±7.6%, p≤0.0001) and in liquid biopsies as well (normal: 82.0±2.0%, adenoma: 80.0±1.7%, carcinoma: 79.8±1.3%, p≤0.01). However, no significant methylation changes were found in inflammatory bowel disease cases. Analyzing microarray data in silico, altered mRNA expression of certain methylation-, and one-carbon metabolism-related genes were detected in tumorous specimens vs. healthy biopsies, from which DNMT1 was upregulated, and folate receptor 2 (FOLR2) was downregulated. DNMT and FOLR2 expression were validated by immunohistochemistry. Furthermore, significantly reduced folic acid and S-adenosylmethionine content were observed in parallel with diminishing 5-methylcytosine levels in adenoma and carcinoma sections compared to normal adjacent to tumor tissue areas by immunolabeling (p≤0.05). Our results suggest that intraindividual monitoring of genome-wide hypomethylation may assist in the recognition of adenoma formation, cancer progression, or remission as well. Moreover, lower global DNA methylation level could be connected to decreased methyl-donor availability with the contribution of reduced FOLR2 expression. Citation Format: Krisztina Andrea Szigeti, Alexandra Kalmár, Gábor Valcz, Barbara Kinga Barták, Zsófia Nagy, Sára Zsigrai, Ildikó Felletár, Árpád V Patai, Tamás Micsik, Márton Papp, Eszter Márkus, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3745.
Crystal Structure of the second bromodomain of human BRD2 in complex with the inhibitor JQ1
Crystal Structure of the first bromodomain of human BRD4 in complex with a diacetylated histone 4 peptide (H4K16acK20ac)
Targeting Low-Druggability Bromodomains: Fragment Based Screening and Inhibitor Design against the BAZ2B Bromodomain
ABSTRACT: Bromodomains are epigenetic reader domains that have recently become popular targets. I... more ABSTRACT: Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable. Bromodomains are epigenetic reader domains within proteins that specifically recognize acetylated lysine (Kac) in histones and other substrate proteins. There are 61 different bromodomains spread across 46 proteins in the human
[1,2,4]Triazolo[4,3‑a]phthalazines: Inhibitors of Diverse
ABSTRACT: Bromodomains are gaining increasing interest as drug targets. Commercially sourced and ... more ABSTRACT: Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]triazolo[4,3-a]phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology. The rapidly expanding field of epigenetics can be broadly divided into two levels of processes: DNA methylation and histone modification. Various post-translational modifications
Background: On most farms the prevalence of mastitis and the number of infected cows is often unk... more Background: On most farms the prevalence of mastitis and the number of infected cows is often unknown. These cases do not show any clinical signs and are identified only by California Mastitis Test (CMT) assessment and Somatic Cell Count (SCC) determination. One of the additional diagnostic methods of mastitis may be involve determination of lipid peroxidation end product in milk. Objective: The aim of the study was to detect the main bacterial pathogens causing mastitis in dairy cows and their impact on lipid peroxidation product, using milk malondialdehyde (MDA) as an indicator of oxidative stress. Methods: During the complex investigation of 123 lactating cows, 612 quarter milk samples were exanimated and classified by clinical examination, abnormal udder secretions, assessment of the CMT, with collecting of milk samples for detection of MDA and bacteriological identification of pathogens causing mastitis. Results: Positive CMT score was recorded of 19.2% (118) exanimated quarters and 12.5% (76) quarters were confirmed bacterial pathogens causing latent (1.3%), subclinical (SM; 8.3%) and clinical (CM; 3.0%) mastitis. The most common bacterial isolates from the mastitis cases were staphylococci (67%) included S. aureus, S. chromogenes, S. haemolyticus and S. warneri. The second most common cause of mastitis were S. uberis and S. faecalis. The concentrations of MDA were significantly higher from SM and CM cases than in milk samples from healthy cows. In addition, we found that quarter milk samples infected with S. uberis were higher MDA levels compared to other pathogens. Conclusion: The higher MDA concentrations in SM and CM milk observed in this study showed that the oxidative stress of infected milk is higher than normal milk. In conclusion, the measurement of milk MDA level could be a potential biomarker for monitoring health status of the mammary gland.
Crystal Structure of the bromodomain of human BAZ2B in complex WITH GSK2801
Crystal Structure of the first bromodomain of human BRD4 in complex with a dihydro-quinazolin ligand
Crystal structure of the second bromodomain of human bromodomain and WD repeat-containing protein 1 isoform A (WDR9)
Crystal Structure of the first bromodomain of human BRD4 in complex with the inhibitor JQ1
Crystal Structure of the fifth bromodomain of human PB1 in complex with salicylic acid
Crystal Structure of the first bromodomain of human BRD4 in complex with a quinazolinone ligand (RVX-OH)
Crystal structure of the second bromodomain of human TAF1L in complex with bromosporine (BSP)
Crystal Structure of the fifth bromodomain of Human Poly-bromodomain containing protein 1 (PB1) in complex with a hydroxyphenyl-propenone ligand
Crystal Structure of the tandem bromodomains of human Transcription initiation factor TFIID subunit 1 (TAF1)
Crystal Structure of the bromodomain of human Transcription activator BRG1 (SMARCA4) in complex with N-Methyl-2-pyrrolidone
Proceedings of the National Academy of Sciences, 2013
Significance Bromo and extraterminal (BET) proteins have diverse roles in regulating tissue-speci... more Significance Bromo and extraterminal (BET) proteins have diverse roles in regulating tissue-specific transcriptional programs, raising safety concerns for their inhibition and suggesting that targeting of specific isoforms or even specific domains within this subfamily is important. We report the discovery and characterization of RVX-208 as a domain-selective inhibitor of BETs and provide a potential mechanism of action of a clinical compound that was identified based on phenotypic screens.
Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery
Molecular BioSystems, 2011
Histone lysine acetylation is a key component of epigenetic regulation of gene transcription. Bro... more Histone lysine acetylation is a key component of epigenetic regulation of gene transcription. Bromodomains, found in histone acetyl transferases and other chromatin-associated proteins, bind selectively to acetylated lysines, acting as "readers" of the histone code, and have recently been shown to contain a druggable binding pocket. Here we report the development of high-throughput assays that quantify the binding of bromodomains to acetylated histone peptides. We have used these assays to screen for histone binding partners of as yet uncharacterized bromodomains, adding to current knowledge of the histone code and expanding the repertoire of assays for chemical probe discovery. We have also demonstrated that these assays can be used to detect small molecule binding from the very weak to the nanomolar range. This assay methodology is thereby anticipated to provide the basis both for broader interactome profiling and for small molecule inhibitor discovery.
Abstract 1690: A histology based approach to spatial single cell analysis of colorectal cancers
Cancer Research, Jun 15, 2022
Background: Single cell sequencing is a powerful tool for the evaluation of intratumoral heteroge... more Background: Single cell sequencing is a powerful tool for the evaluation of intratumoral heterogeneity and the investigation of cancer evolution. Aims: By combining laser microdissection and single cell sequencing, we aimed to link tissue morphology and spatial information with sequencing data of the isolated cells. Materials & methods: In our preliminary study, we used fresh frozen tissue specimen of surgically resected material from a colorectal cancer (CRC) patient containing both cancerous and normal adjacent tissue (NAT). From part of the normal and cancerous tissue exome sequencing was performed in bulk (to assess somatic variants), while the other part was subjected to single cell sequencing. Fresh frozen tissues from both CRC and NAT were cryosectioned at -20°C with section thickness ranging from 16 to 25 µm to ensure that a layer of whole cells are present in the slides. Tissue slides were then scanned using a PANNORAMIC 1000 scanner (3DHISTECH Ltd.). After morphologic evaluation single normal colonocytes and cancerous cells were laser microdissected from the NAT and multiple CRC areas (invasive front, differentiated, non-differentiated cells) by using a CellCut Laser Microdissection system (MMI). The isolated cells were subjected to Repli-G Single Cell WGA Kit (Qiagen) and library preparation followed by whole exome sequencing (WES) on NextSeq 550 (Illumina). Blood sample was also collected before surgical treatment, cell-free DNA was isolated and exome sequencing was completed. Bioinformatic analysis was conducted using BaseSpace and GATK4 best practices. Common and unique variants were identified between cells and also compared with the bulk exom and cell free DNA sequencing results of the patient. Identified variants were further investigated using the oncoKB annotator. Results: Both healthy (1) and cancerous epithelial cells (3) were dissected and sequenced successfully. A median depth of coverage of 192 was achieved with a median of 43.3% of coverage of 50x or above in the target region compared to 73.9 and 52.8% 208 and 97.6% in tissue and plasma samples, respectively. Overall, we identified 105, likely oncogenic” and 2, predicted oncogenic” unique variants in the single cells using the oncoKB annotator. Among the cancerous single cells, we identified 10, likely oncogenic” and 1, predicted oncogenic” common variants (such as ARID4B, DNMT3B, MSH6 and HNF1A), while 15, 25 and 7, likely oncogenic” (such as CTLA4, MLH1, MSH2, CDK12, CDKN1B) variants were identified uniquely in the 3 cancerous cells. Conclusion: We were able to dissect, isolate and sequence single cells from CRC and NAT thus combining valuable morphologic information with sequencing data on a single cellular level with maintained spatial information. The distribution of variants among the single cells shows that it is a viable approach to investigate tumor heterogeneity and to link the morphologic phenotypes and genotypes of cancerous cells. Citation Format: William Jayasekara Kothalawala, Alexandra Kalmár, Gitta Szabó, Barbara Kinga Barták, Sára Zsigrai, Zsófia Brigitta Nagy, Ildikó Felletár, Krisztina Andrea Szigeti, István Takács, Béla Molnár. A histology based approach to spatial single cell analysis of colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1690.
Abstract 3745: Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression
Cancer Research, Jun 15, 2022
Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer t... more Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer types, including colorectal cancer. Abnormality of several factors, such as DNA methyltransferases (DNMT), demethylases, or deviation in methyl-donor (folate and S-adenosylmethionine) availability can contribute to the development of genome-wide hypomethylation. Detection of epigenetic changes as global DNA hypomethylation in cell-free DNA fraction obtained from blood samples can expand the opportunities for the early recognition of colorectal cancer. One of our main goals was the investigation of global DNA methylation patterns in tissue biopsies (n=183) and cell-free DNA fraction of blood samples (n=48) along the colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore possible underlying mechanisms of genome-wide hypomethylation formation in 12 colorectal tumor tissue sections, containing transition zones. Using LINE-1 pyrosequencing, significantly reduced global DNA methylation level was detected in line with cancer progression in tissue specimens (normal: 77.5±1.7%, adenoma: 72.7±4.8%, carcinoma: 69.7±7.6%, p≤0.0001) and in liquid biopsies as well (normal: 82.0±2.0%, adenoma: 80.0±1.7%, carcinoma: 79.8±1.3%, p≤0.01). However, no significant methylation changes were found in inflammatory bowel disease cases. Analyzing microarray data in silico, altered mRNA expression of certain methylation-, and one-carbon metabolism-related genes were detected in tumorous specimens vs. healthy biopsies, from which DNMT1 was upregulated, and folate receptor 2 (FOLR2) was downregulated. DNMT and FOLR2 expression were validated by immunohistochemistry. Furthermore, significantly reduced folic acid and S-adenosylmethionine content were observed in parallel with diminishing 5-methylcytosine levels in adenoma and carcinoma sections compared to normal adjacent to tumor tissue areas by immunolabeling (p≤0.05). Our results suggest that intraindividual monitoring of genome-wide hypomethylation may assist in the recognition of adenoma formation, cancer progression, or remission as well. Moreover, lower global DNA methylation level could be connected to decreased methyl-donor availability with the contribution of reduced FOLR2 expression. Citation Format: Krisztina Andrea Szigeti, Alexandra Kalmár, Gábor Valcz, Barbara Kinga Barták, Zsófia Nagy, Sára Zsigrai, Ildikó Felletár, Árpád V Patai, Tamás Micsik, Márton Papp, Eszter Márkus, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3745.
Crystal Structure of the second bromodomain of human BRD2 in complex with the inhibitor JQ1
Crystal Structure of the first bromodomain of human BRD4 in complex with a diacetylated histone 4 peptide (H4K16acK20ac)
Targeting Low-Druggability Bromodomains: Fragment Based Screening and Inhibitor Design against the BAZ2B Bromodomain
ABSTRACT: Bromodomains are epigenetic reader domains that have recently become popular targets. I... more ABSTRACT: Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable. Bromodomains are epigenetic reader domains within proteins that specifically recognize acetylated lysine (Kac) in histones and other substrate proteins. There are 61 different bromodomains spread across 46 proteins in the human
[1,2,4]Triazolo[4,3‑a]phthalazines: Inhibitors of Diverse
ABSTRACT: Bromodomains are gaining increasing interest as drug targets. Commercially sourced and ... more ABSTRACT: Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]triazolo[4,3-a]phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology. The rapidly expanding field of epigenetics can be broadly divided into two levels of processes: DNA methylation and histone modification. Various post-translational modifications
Background: On most farms the prevalence of mastitis and the number of infected cows is often unk... more Background: On most farms the prevalence of mastitis and the number of infected cows is often unknown. These cases do not show any clinical signs and are identified only by California Mastitis Test (CMT) assessment and Somatic Cell Count (SCC) determination. One of the additional diagnostic methods of mastitis may be involve determination of lipid peroxidation end product in milk. Objective: The aim of the study was to detect the main bacterial pathogens causing mastitis in dairy cows and their impact on lipid peroxidation product, using milk malondialdehyde (MDA) as an indicator of oxidative stress. Methods: During the complex investigation of 123 lactating cows, 612 quarter milk samples were exanimated and classified by clinical examination, abnormal udder secretions, assessment of the CMT, with collecting of milk samples for detection of MDA and bacteriological identification of pathogens causing mastitis. Results: Positive CMT score was recorded of 19.2% (118) exanimated quarters and 12.5% (76) quarters were confirmed bacterial pathogens causing latent (1.3%), subclinical (SM; 8.3%) and clinical (CM; 3.0%) mastitis. The most common bacterial isolates from the mastitis cases were staphylococci (67%) included S. aureus, S. chromogenes, S. haemolyticus and S. warneri. The second most common cause of mastitis were S. uberis and S. faecalis. The concentrations of MDA were significantly higher from SM and CM cases than in milk samples from healthy cows. In addition, we found that quarter milk samples infected with S. uberis were higher MDA levels compared to other pathogens. Conclusion: The higher MDA concentrations in SM and CM milk observed in this study showed that the oxidative stress of infected milk is higher than normal milk. In conclusion, the measurement of milk MDA level could be a potential biomarker for monitoring health status of the mammary gland.
Crystal Structure of the bromodomain of human BAZ2B in complex WITH GSK2801
Crystal Structure of the first bromodomain of human BRD4 in complex with a dihydro-quinazolin ligand
Crystal structure of the second bromodomain of human bromodomain and WD repeat-containing protein 1 isoform A (WDR9)
Crystal Structure of the first bromodomain of human BRD4 in complex with the inhibitor JQ1
Crystal Structure of the fifth bromodomain of human PB1 in complex with salicylic acid
Crystal Structure of the first bromodomain of human BRD4 in complex with a quinazolinone ligand (RVX-OH)
Crystal structure of the second bromodomain of human TAF1L in complex with bromosporine (BSP)
Crystal Structure of the fifth bromodomain of Human Poly-bromodomain containing protein 1 (PB1) in complex with a hydroxyphenyl-propenone ligand
Crystal Structure of the tandem bromodomains of human Transcription initiation factor TFIID subunit 1 (TAF1)
Crystal Structure of the bromodomain of human Transcription activator BRG1 (SMARCA4) in complex with N-Methyl-2-pyrrolidone
Proceedings of the National Academy of Sciences, 2013
Significance Bromo and extraterminal (BET) proteins have diverse roles in regulating tissue-speci... more Significance Bromo and extraterminal (BET) proteins have diverse roles in regulating tissue-specific transcriptional programs, raising safety concerns for their inhibition and suggesting that targeting of specific isoforms or even specific domains within this subfamily is important. We report the discovery and characterization of RVX-208 as a domain-selective inhibitor of BETs and provide a potential mechanism of action of a clinical compound that was identified based on phenotypic screens.
Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery
Molecular BioSystems, 2011
Histone lysine acetylation is a key component of epigenetic regulation of gene transcription. Bro... more Histone lysine acetylation is a key component of epigenetic regulation of gene transcription. Bromodomains, found in histone acetyl transferases and other chromatin-associated proteins, bind selectively to acetylated lysines, acting as "readers" of the histone code, and have recently been shown to contain a druggable binding pocket. Here we report the development of high-throughput assays that quantify the binding of bromodomains to acetylated histone peptides. We have used these assays to screen for histone binding partners of as yet uncharacterized bromodomains, adding to current knowledge of the histone code and expanding the repertoire of assays for chemical probe discovery. We have also demonstrated that these assays can be used to detect small molecule binding from the very weak to the nanomolar range. This assay methodology is thereby anticipated to provide the basis both for broader interactome profiling and for small molecule inhibitor discovery.