Biology subjects

Greenblatt, J. F.

Publications and source records attributed to Greenblatt, J. F..

8 recordsLinked to original sources

bioRxiv2026

Integrative analysis reveals extensive interactions among C2H2 zinc finger proteins at chromatin loop anchors

Cys2-His2 -zinc-finger proteins (C2H2-ZFPs) form the largest class of human transcription factors, yet their potential role (s) in higher-order genome organization has remained largely poorly defined. To date, only a small number of family members, e.g., CTCF and YY1, have been shown to function in chromatin organization. Here, we examined the global relationship between C2H2-ZFPs and long-range chromatin interactions (LRIs). By integrating ChIP-seq datasets for 216 C2H2-ZFPs and genome-wide maps of chromatin looping, we observe that more than 40% of the human C2H2-ZFPs are significantly enriched at LRI anchors. We found that depletion of a subset of such C2H2-ZFPs is associated with altered expression of genes linked by the LRIs they occupy. To investigate whether protein-protein interactions (PPIs) contribute to this pattern, we generated a large-scale interactome using affinity purification followed by mass spectrometry experiments, encompassing 345 C2H2-ZFPs, and LUMIER binary interaction assays for 204 C2H2-ZFPs. We identified 1,732 binary interactions, suggesting extensive connectivity among the C2H2-ZFP family members. Integrative analysis of PPI, ChIP-seq, and chromatin interaction datasets revealed that interacting C2H2-ZFP pairs are significantly co-enriched at LRIs and frequently localize to either the same or opposing loop anchors. Finally, by correlating ChIP-seq with cancer mutational datasets, we observe that DNA-binding sites of [~]35% of LRI-associated C2H2-ZFPs overlap somatic mutations in cancer genomes. Together, our results reveal a widespread network of C2H2-ZFP interactions associated with chromatin loop anchors, providing an important resource for elucidating mechanisms regulating chromatin organization.

genomics↗

bioRxiv2026

ZNF121 recruits YTHDF2 to modulate mRNA stability

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and has been shown to regulate gene expression through the binding of specific reader proteins, such as YTHDF2, which promotes mRNA decay. Previous studies indicate that YTHDF2 has relatively weak intrinsic RNA-binding affinity, suggesting that additional factors may facilitate its association with target transcripts. Here, we show that the C2H2-Zinc Finger protein, ZNF121, binds mRNA in cells and physically interacts with YTHDF2 in the cytoplasm. We demonstrate that approximately 80% of ZNF121-bound mRNAs are also YTHDF2 targets, and that their binding sites highly correlate with each other. Loss of ZNF121 impairs YTHDF2 binding to shared targets and increases their stability, independent of the presence of m6A modifications. Moreover, co-regulated transcripts are enriched for cell-cycle-related pathways, and ZNF121 depletion leads to elevated expression of the oncogene MDM2, implicating ZNF121 in growth control and the DNA damage response. Our findings identify ZNF121 as a cofactor that enhances YTHDF2-mediated mRNA regulation and reveal a previously unknown layer of control in mRNA decay.

molecular biology↗

bioRxiv2025

H3.3 De Novo Mutations Alter Lysine 36 Methylation via Distinct Mechanisms

Bryant-Li-Bhoj syndrome (BLBS) is caused by de novo mutations on histone H3.3 and is generally characterized by severe neurodevelopmental deficits. Oncogenic H3.3 amino acid substitutions were described over the past decade, but the molecular impact of BLBS mutations remained unstudied. The remarkable number and spread of the missense mutations led us to hypothesize that some converge on the same downstream effectors. We recently showed that H3.3G34R/V substitutions, seen in both cancer and BLBS, impair associations with the DNMT3A DNA methyltransferase. Our proteomic, enzymatic, and structural analyses now show that H3.3 BLBS mutations flanking glycine 34 have surprisingly stark effects on H3K36 methyltransferases, drastically altering H3K36 methyl states in cis and the binding of effector proteins with a PWWP domain, including DNMT3A/B. That confirms the existence of molecular commonalities amongst BLBS H3.3 point mutants while providing some of the first mechanistic insights into the syndrome.

molecular biology↗

bioRxiv2024

Recruitment of the m6A/Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48

N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2'-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity. Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO- dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA- binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

molecular biology↗

bioRxiv2023

N-6-methyladenosine (m6A) Promotes the Nuclear Retention of mRNAs with Intact 5' Splice Site Motifs

In humans, misprocessed mRNAs containing intact 5' Splice Site (5'SS) motifs are nuclear retained and targeted for decay by ZFC3H1, a component of the Poly(A) Exosome Targeting complex, and U1-70K, a component of the U1 snRNP. In S. pombe, the ZFC3H1 homolog, Red1, binds to the YTH-domain containing protein Mmi1 to target certain RNA transcripts to nuclear foci for nuclear retention and decay. Here we show that YTHDC1 and YTHDC2, two YTH domain-containing proteins that bind to N-6-methyladenosine (m6A) modified RNAs, interact with ZFC3H1 and U1-70K, and are required for the nuclear retention of mRNAs with intact 5'SS motifs. Disruption of m6A deposition inhibits both the nuclear retention of these transcripts and their accumulation in YTHDC1-enriched foci that are adjacent to nuclear speckles. Endogenous RNAs with intact 5'SS motifs, such as intronic polyadenylated transcripts, tend to be m6A-modified at low levels. Thus, m6A modification acts in a conserved quality control mechanism that targets misprocessed mRNAs for nuclear retention and decay.

molecular biology↗

bioRxiv2020

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links associated with Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion decreased cellular viability and altered the expression of selected targets. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Nucleocapsid protein attenuates stress granule formation and alters gene expression via direct interaction with host mRNAs

The COVID-19 pandemic has caused over one million deaths thus far. There is an urgent need for the development of specific viral therapeutics and a vaccine. SARS-CoV-2 nucleocapsid (N) protein is highly expressed upon infection and is essential for viral replication, making it a promising target for both antiviral drug and vaccine development. Here, starting from a functional proteomics workflow, we initially catalogued the protein-protein interactions of 21 SARS-CoV-2 proteins in HEK293 cells, finding that the stress granule resident proteins G3BP1 and G3BP2 copurify with N with high specificity. We demonstrate that N protein expression in human cells sequesters G3BP1 and G3BP2 through its physical interaction with these proteins, attenuating stress granule (SG) formation. The ectopic expression of G3BP1 in N-expressing cells was sufficient to reverse this phenotype. Since N is an RNA-binding protein, we performed iCLIP-sequencing experiments in cells, with or without exposure to oxidative stress, to identify the host RNAs targeted by N. Our results indicate that SARS-CoV-2 N protein binds directly to thousands of mRNAs under both conditions. Like the G3BPs stress granule proteins, N was found to predominantly bind its target mRNAs in their 3UTRs. RNA sequencing experiments indicated that expression of N results in wide-spread gene expression changes in both unstressed and oxidatively stressed cells. We suggest that N regulates host gene expression by both attenuating stress granules and binding directly to target mRNAs.

molecular biology↗

bioRxiv2019

Identification of a small molecule antagonist for the Tudor domain of SMN

Survival of motor neuron (SMN), a Tudor-domain-containing protein, plays an important role in diverse biological pathways via recognition of symmetrically dimethylated arginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to the motor neuron degenerative disease spinal muscular atrophy (SMA). Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that the sandwich stacking interactions of SMN and compound 1 play a critical role in selective binding to SMN. Various on-target engagement assays support that compound 1 recognizes SMN specifically in a cellular context. In cell studies display that the SMN antagonist prevent the interaction of SMN with R1810me2s of DNA-directed RNA polymerase II subunit POLR2A and results in transcription termination and R-loop accumulation, mimicking depletion of SMN. Thus, in addition to the antisense, RNAi and CRISPR/Cas9 techniques, the potent SMN antagonist could be used as an efficient tool in understanding the biological functions of SMN and molecular etiology in SMA.

biochemistry↗