Eric Milot

The Ubiquitin Carboxyl Hydrolase BAP1 Forms a Ternary Complex with YY1 and HCF-1 and Is a Critical Regulator of Gene Expression

ABSTRACT The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.

Ikaros and GATA-1 Combinatorial Effect Is Required for Silencing of Human γ-Globin Genes

ABSTRACT During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human β-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human γ-gene promoters, thereby contributing to γ-globin gene silencing at the time of the γ- to β-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human γ-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.

Regulation of Neutrophil Survival/Apoptosis by Mcl-1

Neutrophil granulocytes have the shortest lifespan among leukocytes in the circulation and die via apoptosis. At sites of infection or tissue injury, prolongation of neutrophil lifespan is critical for effective host defense. Apoptosis of inflammatory neutrophils and their clearance are critical control points for termination of the inflammatory response. Evasion of neutrophil apoptosis aggravates local injury and leads to persistent tissue damage. The short-lived prosurvival Bcl-2 family protein, Mcl-1 (myeloid cell leukemia-1), is instrumental in controlling apoptosis and consequently neutrophil lifespan in response to rapidly changing environmental cues during inflammation. This paper will focus on multiple levels of control of Mcl-1 expression and function and will discuss targeting Mcl-1 as a potential therapeutic strategy to enhance the resolution of inflammation through accelerating neutrophil apoptosis.

Neuronal ER stress impedes myeloid-cell-induced vascular regeneration through IRE1α degradation of netrin-1.

In stroke and proliferative retinopathy, despite hypoxia driven angiogenesis, delayed revascularization of ischemic tissue aggravates the loss of neuronal function. What hinders vascular regrowth in the ischemic central nervous system remains largely unknown. Using the ischemic retina as a model of neurovascular interaction in the CNS, we provide evidence that the failure of reparative angiogenesis is temporally and spatially associated with endoplasmic reticulum (ER) stress. The canonical ER stress pathways of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme-1α (IRE1α) are activated within hypoxic/ischemic retinal ganglion neurons, initiating a cascade that results in angiostatic signals. Our findings demonstrate that the endoribonuclease IRE1α degrades the classical guidance cue netrin-1. This neuron-derived cue triggers a critical reparative-angiogenic switch in neural macrophage/microglial cells. Degradation of netrin-1, by persistent neuronal ER stress, thereby hinders vascular regeneration. These data identify a neuronal-immune mechanism that directly regulates reparative angiogenesis.

GATA-1 Utilizes Ikaros and Polycomb Repressive Complex 2 To Suppress Hes1 and To Promote Erythropoiesis

ABSTRACT The transcription factor Hairy Enhancer of Split 1 (HES1), a downstream effector of the Notch signaling pathway, is an important regulator of hematopoiesis. Here, we demonstrate that in primary erythroid cells, Hes1 gene expression is transiently repressed around proerythroblast stage of differentiation. Using mouse erythroleukemia cells, we found that the RNA interference (RNAi)-mediated depletion of HES1 enhances erythroid cell differentiation, suggesting that this protein opposes terminal erythroid differentiation. This is also supported by the decreased primary erythroid cell differentiation upon HES1 upregulation in Ikaros-deficient mice. A comprehensive analysis led us to determine that Ikaros favors Hes1 repression in erythroid cells by facilitating recruitment of the master regulator of erythropoiesis GATA-1 alongside FOG-1, which mediates Hes1 repression. GATA-1 is then necessary for the chromatin binding of the NuRD remodeling complex ATPase MI-2, the transcription factor GFI1B, and the histone H3K27 methyltransferase EZH2 along with Polycomb repressive complex 2. We show that EZH2 is required for the transient repression of Hes1 in erythroid cells. In aggregate, our results describe a mechanism whereby GATA-1 utilizes Ikaros and Polycomb repressive complex 2 to promote Hes1 repression as an important step in erythroid cell differentiation.

The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer

Background: The relevance of ASXL2 to the function of the histone H2A deubiquitinase BAP1 remains unknown. Results: ASXL2 promotes the assembly by BAP1 of a composite ubiquitin-binding interface (CUBI) required for DUB activity and coordination of cell proliferation. Conclusion: Cancer-associated mutations of BAP1 disrupt BAP1-ASXL2 interaction and function. Significance: We provide novel insights into BAP1 tumor suppressor function. The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A Lys-119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1, protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1, and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination-promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1, which generate a composite ubiquitin-binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving (i) the ubiquitin carboxyl hydrolase catalytic domain of BAP1, which interacts with the hydrophobic patch of ubiquitin, and (ii) the CTD domain, which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI and notably an in-frame deletion in the CTD that inhibits its interaction with ASXL1/2 and DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of the BAP1/ASXL2 axis might contribute to cancer development.

Ikaros interacts with P-TEFb and cooperates with GATA-1 to enhance transcription elongation

Ikaros is associated with both gene transcriptional activation and repression in lymphocytes. Ikaros acts also as repressor of human γ-globin (huγ-) gene transcription in fetal and adult erythroid cells. Whether and eventually, how Ikaros can function as a transcriptional activator in erythroid cells remains poorly understood. Results presented herein demonstrate that Ikaros is a developmental-specific activator of huγ-gene expression in yolk sac erythroid cells. Molecular analysis in primary cells revealed that Ikaros interacts with Gata-1 and favors Brg1 recruitment to the human β-globin Locus Control Region and the huγ-promoters, supporting long-range chromatin interactions between these regions. Additionally, we demonstrate that Ikaros contributes to transcription initiation and elongation of the huγ-genes, since it is not only required for TBP and RNA Polymerase II (Pol II) assembly at the huγ-promoters but also for conversion of Pol II into the elongation-competent phosphorylated form. In agreement with the latter, we show that Ikaros interacts with Cyclin-dependent kinase 9 (Cdk9), which contributes to efficient transcription elongation by phosphorylating the C-terminal domain of the large subunit of Pol II on Serine 2, and favours Cdk9 recruitment to huγ-promoters. Our results show that Ikaros exerts dual functionality during gene activation, by promoting efficient transcription initiation and elongation.

Acetylation of EKLF Is Essential for Epigenetic Modification and Transcriptional Activation of the β-Globin Locus

ABSTRACT Posttranslational modifications of transcription factors provide alternate protein interaction platforms that lead to varied downstream effects. We have investigated how the acetylation of EKLF plays a role in its ability to alter the β-like globin locus chromatin structure and activate transcription of the adult β-globin gene. By establishing an EKLF-null erythroid line whose closed β-locus chromatin structure and silent β-globin gene status can be rescued by retroviral infection of EKLF, we demonstrate the importance of EKLF acetylation at lysine 288 in the recruitment of CBP to the locus, modification of histone H3, occupancy by EKLF, opening of the chromatin structure, and transcription of adult β-globin. We also find that EKLF helps to coordinate this process by the specific association of its zinc finger domain with the histone H3 amino terminus. Although EKLF interacts equally well with H3.1 and H3.3, we find that only H3.3 is enriched at the adult β-globin promoter. These data emphasize the critical nature of lysine acetylation in transcription factor activity and enable us to propose a model of how modified EKLF integrates coactivators, chromatin remodelers, and nucleosomal components to alter epigenetic chromatin structure and stimulate transcription.

The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis.

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of IkNULL hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.

Role for Myeloid Nuclear Differentiation Antigen in the Regulation of Neutrophil Apoptosis during Sepsis

RATIONALE Suppressed neutrophil apoptosis, a hallmark of sepsis, perpetuates inflammation and delays resolution. Myeloid nuclear differentiation antigen (MNDA) is expressed only in myeloid cells and has been implicated in cell differentiation; however, its function in mature neutrophils is not known. OBJECTIVES We studied whether MNDA could contribute to regulation of apoptosis of neutrophils from healthy subjects and patients with sepsis, and investigated the impact of MNDA knockdown on apoptosis. METHODS Human neutrophils were challenged with mediators of sepsis and neutrophils from patients with sepsis were cultured to investigate cleavage and cytoplasmic accumulation of MNDA. MNDA was knocked down in myeloid HL-60 cells to investigate development of apoptosis. MEASUREMENTS AND MAIN RESULTS During constitutive apoptosis of human neutrophils, MNDA is cleaved by caspases and accumulated in the cytoplasm, where it promotes degradation of the antiapoptotic protein Mcl-1, thereby accelerating collapse of mitochondrial transmembrane potential. Culture of neutrophils with LPS, bacterial DNA, or platelet-activating factor prevented MNDA cleavage and cytoplasmic accumulation. MNDA knockdown with short hairpin RNA markedly attenuated Mcl-1 turnover and conferred resistance to stress-induced apoptosis in HL-60 cells. Neutrophils from patients with severe sepsis exhibited markedly suppressed apoptosis that was associated with impaired cytoplasmic MNDA accumulation, preservation of Mcl-1 expression, and mitochondrial transmembrane potential. Culture of neutrophils of healthy subjects with septic plasma delayed apoptosis and cytoplasmic MNDA accumulation. CONCLUSIONS These results indicate that cytoplasmic accumulation of MNDA facilitates progression of apoptosis and suggest that impaired cytoplasmic MNDA accumulation contributes to delayed neutrophil apoptosis in patients with severe sepsis.