NuRD Chromatin Remodeling Complex Research Articles

Defective Production, Turnover, and Secretion of the Platelet α-Granule Protein P-Selectin In Mice with Disrupted FOG1-NuRD Interaction

AbstractAbstract 547We reported recently that point mutations in the GATA-1 co-factor FOG-1 that disrupt binding to the NuRD chromatin remodeling complex display defects in the erythroid and megakaryocyte (MK) lineages. Homozygous FOG-1 mutant (ki/ki) mice display a Gray Platelet Syndrome (GPS)-like macrothrombocytopenia. Specifically, ki/ki platelets exhibit a paucity of α-granules and increased numbers of lysosomal-like vacuoles, reminiscent of the platelet morphology observed in human patients with GPS due to GATA-1 mutations. To understand the molecular basis for the phenotype in ki/ki platelets, we compared mRNA and protein expression of α-granule proteins in wildtype (wt) and ki/ki MK. P-selectin mRNA in ki/ki MKs was 3–5 fold reduced when compared to controls, while platelet factor 4 (Pf4), platelet basic protein (PBP) and von Willebrand factor (Vwf), were only mildly reduced. Strikingly, P-selectin protein levels were more severely reduced in ki/ki MK and platelets being nearly undetectable by Western blot and immunohistochemistry. In contrast PF4, PBP, and vWF protein levels were normal. P-selectin loss was MK-specific as it was expressed at normal levels in endothelial cells. The dramatic decrease in P-selectin protein in ki/ki MK relative to the more moderate reduction in mRNA was due to a >5-fold decrease in protein half-life, as measured by metabolic pulse-chase and immunoprecipitation experiments. This suggests that P-selectin in ki/ki MKs is largely mistargeted to lysosomes. Nevertheless, immunofluorescence microscopy and volume de-convolution analyses of ki/ki platelets showed that the remaining P-selectin resided in vesicular compartments that were distinct from Lamp1 (a lysosomal marker)-containing granules, indicating that a fraction of P-selectin remains within α-like granules. Interestingly, the reduction in P-selectin levels in ki/ki platelets was accompanied by a defect in agonist-stimulated degranulation. While stimulation of wt platelets with AYP, thrombin or phrobol myristate acetate induced α-granule, dense granule and lysosome degranulation, ki/ki platelets failed to do so or undergo platelet aggregation. In summary, the GPS-like phenotype in FOG-1/NuRD interaction-defective MKs involves reduced P-selectin mRNA production, accelerated turnover of P-selectin, abnormal granule formation, and defective degranulation in response to diverse agonists. These findings may partially explain patients having platelet α-granule deficiency associated with decreased P-selectin and selective impairment of thrombin-induced activation with GPS. Future work will determine whether similar defects occur in human GPS. Disclosures:No relevant conflicts of interest to declare.

Foxp1/2/4-NuRD Interactions Regulate Gene Expression and Epithelial Injury Response in the Lung via Regulation of Interleukin-6

To determine the underlying mechanism of Foxp1/2/4-mediated transcriptional repression, a yeast two-hybrid screen was performed that identified p66beta, a transcriptional repressor and component of the NuRD chromatin-remodeling complex. We show that direct interactions between Foxp1/4 and p66beta are mediated by the CR2 domain within p66beta and the zinc finger/leucine zipper repression domain found in Foxp1/2/4. These direct interactions are functionally relevant as overexpression of p66beta in combination with Foxp factors cooperatively represses Foxp target gene expression, whereas loss of p66 and Foxp factors results in de-repression of endogenous Foxp target genes in lung epithelial cells. Moreover, the NuRD components HDAC1/2 associate in a macromolecular complex with Foxp proteins, and loss of expression or inhibition of HDAC1/2 activity leads to de-repression of Foxp target gene expression. Importantly, we show in vivo that Foxp1 and HDAC2 act cooperatively to regulate expression of the cytoprotective cytokine interleukin-6, which results in increased resistance to hyperoxic lung injury in Foxp1/HDAC2 compound mutant animals. These data reveal an important interaction between the Foxp transcription factors and the NuRD chromatin-remodeling complex that modulates transcriptional repression critical for the lung epithelial injury response.

The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

By stimulating collagen synthesis and myofibroblasts differentiation, transforming growth factor-β (TGF- β) plays a pivotal role in tissue repair and fibrosis. The early growth response-1 (Egr-1) transcription factor mediates profibrotic TGF-β responses, and its expression is elevated in biopsies from patients with scleroderma. NGF1-A-binding protein 2 (Nab2) is a conserved transcriptional cofactor that directly binds to Egr-1 and positively or negatively modulates Egr-1 target gene transcription. Despite the recognized importance of Nab2 in governing the intensity of Egr-1-dependent responses, the regulation and function of Nab2 in the context of fibrotic TGF-β signaling is unknown. Here we show that TGF-β caused a time-dependent stimulation of Nab2 protein and mRNA in normal fibroblasts. Ectopic expression of Nab2 in these cells blocked Egr-1-dependent transcriptional responses, and abrogated TGF-β-induced stimulation of collagen synthesis and myofibroblasts differentiation. These inhibitory effects of Nab2 involved recruitment of the NuRD chromatin remodeling complex to the COL1A2 promoter and were accompanied by reduced histone H4 acetylation. Mice with targeted deletion of Nab2 displayed increased collagen accumulation in the dermis, and genetic or siRNA-mediated loss of Nab2 in fibroblasts was associated with constitutively elevated collagen synthesis and accentuation of Egr-1-dependent TGF-β responses in vitro. Expression of Nab2 was markedly up-regulated in skin biopsies from patients with scleroderma, and was localized primarily to epidermal keratinocytes. In contrast, little Nab2 could be detected in dermal fibroblasts. These results identify Nab2 as a novel endogenous negative regulator of Egr-1-dependent TGF-β signaling responsible for setting the intensity of fibrotic responses. Defective Nab2 expression or function in dermal fibroblasts might play a role in persistent fibrotic responses in scleroderma.

RCS1, a substrate of APC/C, controls the metaphase to anaphase transition

The anaphase-promoting complex/cyclosome (APC/C) controls the onset of anaphase by targeting securin for destruction. We report here the identification and characterization of a substrate of APC/C, RCS1, as a mitotic regulator that controls the metaphase-to-anaphase transition. We showed that the levels of RCS1 fluctuate in the cell cycle, peaking in mitosis and dropping drastically as cells exit into G(1). Indeed, RCS1 is efficiently ubiquitinated by APC/C in vitro and degraded during mitotic exit in a Cdh1-dependent manner in vivo. APC/C recognizes a unique D-box at the N terminus of RCS1, as mutations of this D-box abolished ubiquitination in vitro and stabilized the mutant protein in vivo. RCS1 controls the timing of the anaphase onset, because the loss of RCS1 resulted in a faster progression from the metaphase to anaphase and accelerated degradation of securin and cyclin B. Biochemically, mitotic RCS1 associates with the NuRD chromatin-remodeling complex, and this RCS1 complex is likely involved in regulating gene expression or chromatin structure, which in turn may control anaphase onset. Our study uncovers a complex regulatory network for the metaphase-to-anaphase transition.

Members of the NuRD Chromatin Remodeling Complex Interact with AUF1 in Developing Cortical Neurons

Chromatin remodeling plays an important role in coordinating gene expression during cortical development, however the identity of molecular complexes present in differentiating cortical neurons that mediate the process remains poorly understood. The A + U-rich element-binding factor 1 (AUF1) is a known regulator of messenger RNA stability and also acts as a transcription factor upon binding to AT-rich DNA elements. Here we show that AUF1 is specifically expressed in subsets of proliferating neural precursors and differentiating postmitotic neurons of the developing cerebral cortex. Moreover, AUF1 is coexpressed with histone deacetylase 1 (HDAC1) and metastasis-associated protein 2 (MTA2), members of the nucleosome remodeling and histone deacetylase complex. AUF1 specifically and simultaneously binds to HDAC1, MTA2, and AT-rich DNA element, its gene regulatory function is modulated by the extent of histone acetylation and in animals lacking AUF1, the composition of the complex is modified. These results suggest that AUF1 is involved in integrating genetic and epigenetic signals during cortical development through recruiting HDAC1 and MTA2 to AT-rich DNA elements.

The Transcription Factor Snail Mediates Epithelial to Mesenchymal Transitions by Repression of Estrogen Receptor-α

The estrogen receptor (ER)-alpha (ESR1) is a key regulatory molecule in mammary epithelial cell development. Loss of ER-alpha in breast cancer is correlated with poor prognosis, increased recurrence after treatment, and an elevated incidence of metastasis. A proposed molecular pathway by which ER-alpha acts to constrain invasive growth in breast cancer cells involves direct, ER-alpha-dependent expression of metastasis-associated protein 3, a cell-type-specific component of the Mi-2/NuRD chromatin remodeling complex. MTA3 in turn represses expression of Snail, a transcription factor linked to epithelial to mesenchymal transition and cancer metastasis. To elucidate its role(s) in epithelial to mesenchymal transition (EMT), we expressed Snail in the noninvasive, ER-alpha-positive MCF-7 cell line. Snail expression led to decreased cell-cell adhesion and increased cell invasiveness. Furthermore, we observed loss of ER-alpha expression at both the RNA and protein level that was accompanied by direct interaction of Snail with regulatory DNA sequences at the ESR1 locus. A consequence of loss of ER-alpha function in this system was the increased abundance of key components of the TGF-beta signaling pathway. Thus, cross-talk among ER-alpha, Snail, and the TGF-beta pathway appears to control critical phenotypic properties of breast cancer cells.