Biology subjects

Klein, D. C.

Publications and source records attributed to Klein, D. C..

3 recordsLinked to original sources

bioRxiv2025

The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

SWI/SNF chromatin remodeling complexes regulate gene expression by modulating nucleosome positioning, yet their roles in metal homeostasis during skeletal muscle development remain unclear. Here, we uncover distinct functions of the BAF, PBAF, and ncBAF complexes in myoblast proliferation under metal stress. While knockdown (KD) of Baf250a (BAF-specific) or Brd9 (ncBAF-specific) reduces myoblast proliferation, Baf180 (PBAF-specific) KD does not impair cell proliferation under basal conditions. Interestingly, supplementation with copper (Cu) or zinc (Zn) rescues proliferation in Baf250a- and Brd9-deficient myoblasts but paradoxically inhibits growth in Baf180 KD cells. Mechanistically, Baf180 KD disrupts Cu and Zn homeostasis, leading to intracellular Cu accumulation without labile Cu pools and impaired expression of Atp7a, a key Cu exporter. Transcriptomic analyses reveal widespread gene dysregulation in metal-treated Baf180-deficient cells, while metal supplementation promotes pro-proliferative gene expression in Baf250a- and Brd9-KD myoblasts. CUT&RUN assays demonstrate that metal-responsive transcription factor Mtf1 exhibits increased chromatin binding upon Cu treatment, targeting genes involved in stress response and myogenesis. Notably, Mtf1 colocalizes with Baf180 in the nucleus and co-immunoprecipitates with both conserved SWI/SNF subunits and Baf180, suggesting a functional interplay between PBAF and Mtf1 in regulating metal-dependent gene expression. Our findings establish the PBAF complex as a crucial regulator of Cu/Zn homeostasis in myoblast proliferation via Mtf1, while metal supplementation compensates for BAF and ncBAF dysfunction but exacerbates defects in PBAF-deficient cells. This study reveals a novel link between chromatin remodeling, metal signaling, and muscle development, with implications for stress adaptation and metabolic regulation in myogenesis.

molecular biology↗

bioRxiv2024

Cysteine Rich Intestinal Protein 2 is a copper-Responsive regulator of skeletal muscle differentiation.

Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.

molecular biology↗

bioRxiv2021

FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells

The FACT complex is a conserved histone chaperone with essential roles in transcription and histone deposition. FACT is essential in pluripotent and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block induction of pluripotent stem cells, yet the mechanism through which FACT regulates cell fate decisions remains unclear. To determine this mechanism, we used inducible depletion of FACT subunit SPT16 in murine embryonic stem cells paired with genomic factor localization, nascent transcription, and chromatin accessibility analyses. Over a timecourse of SPT16 depletion, nucleosomes invade loci bound by master pluripotency factors and gene-distal DNaseI hypersensitive sites. Simultaneously, transcription of Pou5f1 (OCT4), Sox2, Nanog, and enhancer RNAs produced at the genes associated enhancers are downregulated, suggesting that FACT regulates expression of the pluripotency factors themselves. We find that FACT maintains cellular pluripotency through a precise nucleosome-based regulatory mechanism for appropriate expression of both coding and non-coding transcripts associated with pluripotency.

molecular biology↗