Zinc-finger Repressor Protein Research Articles

Cholinergic deficits selectively boost cortical intratelencephalic control of striatum in male Huntington’s disease model mice

Huntington’s disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/− knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/− HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.

Meso scale discovery-based assays for the detection of aggregated huntingtin.

Huntington’s disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1–97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.

Genetics and Epigenetics of Fetal Hemoglobin Control

Expression of fetal hemoglobin (HbF, α2γ2) greatly ameliorates the severity of the major hemoglobin disorders, sickle cell disease (SCD), and the β-thalassemias. Efforts to reactivate HbF in adults with these disorders have relied on empirical observations or therapeutic modalities that are indirect. A major goal for the field is the development of targeted reactivation of HbF through relief of γ-globin gene silencing. The regulatory factors that participate in the switch from HbF to HbA in ontogeny and in γ-gene silencing in the adult have been elusive, therefore precluding mechanism-based reactivation of HbF. Recent findings, largely derived from genome-wide association studies (GWAS), have transformed the current understanding of globin switching. This presentation will review recent evidence supporting direct involvement of the zinc-finger repressor protein BCL11A in both developmental switching of globins and HbF silencing in the adult. These studies include the impact of naturally occurring genetic variation at the BCL11A locus on HbF levels, proof-of-principle experiments in genetically engineered mice suggesting that interference with BCL11A action alone may be sufficient to provide therapeutic elevation of HbF, and the nature of protein partners of BCL11A that may mediate some aspects of BCL11A function. Recent findings on the manner in which genetic variation within the BCL11A locus influences BCL11A expression provide special insight into quantitative aspects of HbF regulation and raise the possibility of new strategies to cripple BCL11A. The opportunities and challenges for the development of mechanism-based reactivation of HbF will be discussed in the context of ongoing efforts to exploit small molecule and genetic approaches. The tools are in hand to translate an improved understanding of globin gene regulation for the benefit of patients with the major hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

ID1 Facilitates the Growth and Metastasis of Non-Small Cell Lung Cancer in Response to Nicotinic Acetylcholine Receptor and Epidermal Growth Factor Receptor Signaling

Expression of ID1 (inhibitor of differentiation) has been correlated with the progression of a variety of cancers, but little information is available on its role in non-small cell lung cancer (NSCLC). Here we show that ID1 is induced by nicotinic acetylcholine receptor (nAChR) and epidermal growth factor receptor (EGFR) signaling in a panel of NSCLC cell lines and primary cells from the lung. ID1 induction was Src dependent and mediated through the α7 subunit of nAChR; transfection of K-Ras or EGFR to primary cells induced ID1. ID1 depletion prevented nicotine- and EGF-induced proliferation, migration, and invasion of NSCLC cells and angiogenic tubule formation of human microvascular endothelial cells from lungs (HMEC-Ls). ID1 could induce the expression of mesenchymal markers such as vimentin and fibronectin by downregulating ZBP-89, a zinc finger repressor protein. ID1 levels were elevated in tumors from mice that were exposed to nicotine. Further, human lung tissue microarrays (TMAs) showed elevated levels of ID1 in NSCLC samples, with maximal levels in metastatic lung cancers. Quantitative reverse transcription-PCR (RT-PCR) performed on patient lung tumors showed that ID1 levels were elevated in advanced stages of NSCLC and correlated with elevated expression of vimentin and fibronectin, irrespective of smoking history.

Control of Hemoglobin Switching by BCL11A.

Abstract 5All vertebrates switch expression of globin chains during development. In humans b-like globins switch from embryonic to fetal to adult, whereas in the mouse a single switch from embryonic to adult occurs. The switch from human fetal (g) to adult (b) expression is especially critical in the b-hemoglobin disorders, such as sickle cell anemia and the b-thalassemias. Delay of the switch or reactivation of the fetal gene in the adult stage greatly ameliorates clinical severity. Despite intensive molecular studies of the human b-globin cluster over more than two decades, the proteins regulating the switch, and the mechanisms controlling the process, have been largely elusive. Recently, genome-wide association studies identified genetic variation at a chromosome 2 locus that correlates with the level of HbF in different populations. The most highly associated single nucleotide polymorphisms (SNPs) reside in an intron of the BCL11A gene, which encodes a zinc-finger repressor protein. Previously we showed that shRNA-mediated ex vivo knockdown of BCL11A in cultured human CD34-derived erythroid precursors leads to robust HbF expression, consistent with a role for BCL11A in maintaining g-genes in a silenced state in adult cells. To address in vivo roles of BCL11A either in development or in globin gene silencing in an intact individual, we have employed stringent genetic tests of function in mice that carry a complete human b-globin gene cluster as a yeast artificial chromosome transgene (b-locus mice). Knockout of BCL11A in mice leads to failure to silence the endogenous b-like embryonic genes in adult erythroid cells of the fetal liver (>2500-fold derepression). The ratio of human g to b globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that g constitutes >90% of the b-like human expression at embryonic day (E)14.5 and >75% at E18.5. These quantitatively striking findings indicate that BCL11A controls developmental silencing of g-globin gene expression. To address by formal genetics the contribution of BCL11A to g silencing in adult animals we have employed conditional inactivation of BCL11A through hematopoietic- and erythroid-specific Cre-alleles. These experiments reveal that BCL11A is also required in vivo for g-gene silencing in adults. We observed that human g-globin expression is persistently derepressed >2000-fold (as compared to littermate controls) in bone marrow erythroblasts of 15-20 week old b-locus mice upon erythroid-specific deletion of BCL11A. Taken together, these findings establish BCL11A as the first genetically validated transcriptional regulator of both developmental control of globin switching and silencing of g-globin expression in adults. The recognition of these roles for BCL11A now permits focused mechanistic studies of the switch. In human erythroid cells, BCL11A physically interacts with at least two corepressor complexes, Mi-2/NuRD and LSD1/CoREST, as well as the erythroid transcription factor GATA-1 and the HMG-box protein SOX6. Rather than binding to the promoters of the g- or b-globin genes as do these latter factors, BCL11A protein occupies the upstream locus control and g-d-intergenic regions of the b-globin cluster (as determined by high resolution ChIP-Chip analysis), suggesting that BCL11A mediates long-range interactions and/or reconfigures the locus during different stages. An in-depth mechanistic understanding of globin switching offers the prospect for design of target-based activation of HbF in adult erythroid cells of patients with hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

RelB NF-κB Represses Estrogen Receptor α Expression via Induction of the Zinc Finger Protein Blimp1

Aberrant constitutive expression of NF-kappaB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERalpha)-negative breast cancers versus ERalpha-positive ones, due in part to repression of RelB synthesis by ERalpha signaling. Notably, RelB promoted a more invasive phenotype in ERalpha-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERalpha synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERalpha (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERalpha-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-kappaB subunit mediates repression, specifically of ERalpha, thereby promoting a more migratory phenotype.

Neuron restrictive silencer factor as a modulator of neuropeptide gene expression

We hypothesize that the transcription factor neuron restrictive silencer factor (NRSF) is an important determinant of the expression of the preprotachykinin (PPTA) gene (encoding substance P and Neurokinin A) and arginine vasopressin (AVP) both in neuronal and nonneuronal cells. NRSF, a zinc finger repressor protein, binds the NRSE motif found in many neuronal specific genes at a variety of promoter locations. However, it is found in a similar location at the major transcriptional start site, within both PPTA and AVP peptide promoters. We have correlated modulation of NRSF activity with expression of AVP and PPTA in a variety of cell types, indicating the general mechanism by which this protein may regulate expression. Specifically, they are as follows:(1). Expression of NRSF dramatically represses PPTA promoter activity in reporter gene constructs in primary cultures of DRG neurons.(2). The PPTA promoter activity is regulated differentially in osteoarthritic compared to normal chondrocytes. This regulation correlates with the region containing the NRSE site.(3). We have correlated a splice variant of NRSF with the establishment and progression of small cell lung carcinoma (SCLC) and demonstrated that NRSF variants can directly affect the activity of the AVP promoter in reporter gene constructs. If the deregulated expression of peptides in these diseases point to the mechanism determining the pathology, then perhaps targeting protocols that correct this deregulation may also reverse the specific disease phenotypes. Our data would indicate that modulation of NRSF activity would be a target for such intervention.

Regulation of Drosophila Adh promoter switching by an initiator-targeted repression mechanism.

The stage-specific expression of the Drosophila alcohol dehydrogenase (Adh) gene is achieved through the alternate activation of two tandem promoters. The proximal promoter is active primarily during late embryonic development and early larval stages, while the distal promoter is active in late third instar larvae and adults. Here, we provide evidence that this Adh promoter switch is regulated by a zinc finger repressor protein (AEF-1) that is expressed predominantly in adult flies and targets the initiator region of the proximal promoter. We propose that AEF-1 plays a critical role in Adh promoter switching by blocking interactions between a component of the general transcription machinery and the initiator region of the proximal promoter.

MIG1 overexpression causes flocculation in Saccharomyces cerevisiae.

MIG1, encoding a C2H2 zinc-finger repressor protein involved in carbon catabolite repression, was found to play a role in non-sexual flocculation of Saccharomyces cerevisiae. Disruption of MIG1 in a flocculent mutant strain of NCYC 227, resulted in a non-flocculent phenotype. Expression of MIG1 on a 2 mu pRS426 vector in a non-flocculent strain, YM 4134, caused flocculation; MIG1 on a high-copy-number LEU2-d plasmid caused intense flocculation in the same strain. Mutations in the SSN6 and TUP1 genes confer a flocculent phenotype in non-flocculent strains of S. cerevisiae, and it has been shown that Mig1 can tether the Ssn6p-Tup1p complex to the regulatory regions of glucose-repressible genes. Mutations in tup1 in a MIG1 background caused flocculation while double mutants of TUP1 and MIG1 did not flocculate. Based on these results, a model for the role of MIG1 in flocculation gene regulation is proposed.

Developmentally Regulated Mouse Gene NK10 Encodes a Zinc Finger Repressor Protein with Differential DNA-Binding Domains

Using oligonucleotides complementary to the conserved inter-finger region of a variety of previously described zinc finger-encoding genes, a novel mouse gene was cloned and characterized. The gene is localized on chromosome 8 and comprises five exons. Its corresponding mRNA is developmentally regulated in various tissues and includes an open reading frame encoding a protein of 72,422 daltons. It shares amino-terminal homologies with human KRAB (or FPB) boxes, and contains 13 zinc fingers of the C2-H2 type. The NK10 KRAB domains exhibit repressing activity when tested in GAL4 fusion protein assays. Cloning of putative target sequences revealed that the individual domains differentially contribute to zinc-dependent target DNA binding.