Biology subjects

Rais, Y.

Publications and source records attributed to Rais, Y..

5 recordsLinked to original sources

bioRxiv2024

Identification and quantification of human relaxin proteins by immunoaffinity-mass spectrometry

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein), and a broader tissue specificity RLN2 (REL2_HUMAN). Due to their structural similarity, REL1 and REL2 proteins were collectively named a human relaxin protein in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays could reveal the identity and concentration of REL1 and REL2 in biological samples and facilitate evaluation of these proteins for diagnostic applications. RT-PCR revealed the high levels of RLN1 and RLN2 transcripts in prostate and breast cancer cell lines. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in numerous biological samples. IA-SRM assay of REL2 revealed its undetectable levels (<9 pg/mL) in control female and male sera, relatively high levels of REL2 in maternal sera (median 331 pg/mL, 120 patients), and a biphasic expression of REL2 across the gestational weeks. IA-SRM assays discovered potential cross-reactivity and false-positive measurements for relaxin immunoassays. The developed IA-SRM assays will facilitate investigation of physiological and pathological roles of REL1 and REL2 peptide hormones.

biochemistry↗

bioRxiv2022

Histone exchange sensors reveal variant specific dynamics in mouse embryonic stem cells

Eviction of histones from nucleosomes and their exchange with newly synthesized or alternative variants is a central epigenetic determinant. Here, we define the genome-wide incorporation and exchange pattern of canonical and non-canonical histone variants in mouse embryonic stem cells by implementing a recently established, genetically encoded exchange sensor. While exchange of all measured variants scales with transcription, we describe variant-specific associations with transcription elongation and Polycomb binding. We found considerable exchange of H3.1 and H2B variants in heterochromatin and repeat elements, contrasting the stable incorporation and little exchange of H3.3 in these regions. This unexpected association between H3.3 incorporation and exchange of canonical variants is also evident in active promoters and enhancers, and further validated by reduced H3.1 dynamics following depletion of the HIRA H3.3-specific chaperone. The sensor system provides a powerful tool for studying regulation of histone dynamics toward understanding its role in shaping the epigenetic landscape in vivo.

genomics↗

bioRxiv2020

Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein with Orthogonal Immunoprecipitation-Mass Spectrometry Assays

TMPRSS2-ERG gene fusion, a molecular alteration driving nearly a half of prostate cancer cases, has been intensively characterized at the transcript level, while limited studies explored the molecular identity and function of the endogenous fusion at the protein level. Here, we developed and applied immunoprecipitation-mass spectrometry (IP-MS) assays for the measurement of a low-abundance T1E4 TMPRSS2-ERG fusion protein, its isoforms and its interactome in VCaP prostate cancer cells. IP-MS assays quantified total ERG ([~]27,000 copies/cell) and its four unique isoforms, and revealed that the T1E4-ERG isoform accounts for 71% of the total ERG protein in VCaP cells. For the first time, the N-terminal peptide (methionine-truncated and N-acetylated TASSSSDYGQTSK) unique for the T1/E4 fusion was identified and quantified. IP-MS with the C-terminal antibodies identified 29 proteins in the ERG interactome, including SWI/SNF chromatin remodeling complex subunits and numerous transcriptional co-regulators. Our data also suggested that TMPRSS2-ERG protein-protein interactions were exerted through at least two different regions. Knowledge on the distinct TMPRSS2-ERG protein isoforms and interactomes may facilitate development of more accurate diagnostics and targeted therapeutics of prostate cancer.

molecular biology↗

bioRxiv2018

Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency

The Nucleosome Remodeling and Deacytelase (NuRD) complex is a co-repressive complex involved in many pathological and physiological processes in the cell. Previous studies have identified one of its components, Mbd3, as a potent inhibitor for reprogramming of somatic cells to pluripotency. Following OSKM induction, early and partial depletion of Mbd3 protein followed by applying naive ground-state pluripotency conditions, results in a highly efficient and near-deterministic generation of mouse iPS cells. Increasing evidence indicates that the NuRD complex assumes multiple mutually exclusive protein complexes, and it remains unclear whether the deterministic iPSC phenotype is the result of a specific NuRD sub complex. Since complete ablation of Mbd3 blocks somatic cell proliferation, here we aimed to identify alternative ways to block Mbd3-dependent NuRD activity by identifying additional functionally relevant components of the Mbd3/NuRD complex during early stages of reprogramming. We identified Gatad2a (also known as P66), a relatively uncharacterized NuRD-specific subunit, whose complete deletion does not impact somatic cell proliferation, yet specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuit during both stem cell differentiation and reprogramming to pluripotency. Complete ablation of Gatad2a in somatic cells, but not Gatad2b, results in a deterministic naive iPSC reprogramming where up to 100% of donor somatic cells successfully complete the process within 8 days. Genetic and biochemical analysis established a distinct sub-complex within the NuRD complex (Gatad2a-Chd4-Mbd3) as the functional and biochemical axis blocking reestablishment of murine naive pluripotency. Disassembly of this axis by depletion of Gatad2a, results in resistance to conditions promoting exit of naive pluripotency and delays differentiation. We further highlight context- and posttranslational dependent modifications of the NuRD complex affecting its interactions and assembly in different cell states. Collectively, our work unveils the distinct functionality, composition and interactions of Gatad2a-Chd4-Mbd3/NuRD subcomplex during the resolution and establishment of mouse naive pluripotency.

developmental biology↗

bioRxiv2017

High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM)1 has provoked interest to define the molecular characteristics of this process2-7. Despite important progress, the dynamics of epigenetic reprogramming at high resolution in correctly reprogrammed iPSCs and throughout the entire process remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using a highly efficient deterministic murine reprogramming system engineered through optimized inhibition of Gatad2a-Mbd3/NuRD repressive sub-complex. This comprehensive characterization provides single-day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. CAPGs are predominantly regulated by Myc, and are enriched for cell biosynthetic regulatory functions. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis demonstrated that de novo DNA methylation is not required for highly efficient conducive iPSC reprogramming, and identified a group of super-enhancers targeted by OSK, whose early demethylation marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are distinctively regulated by multiple synergystic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naive pluripotency.

developmental biology↗