Cell Type-specific Regulation Research Articles

Cell type-specific transcriptional programs in mouse prefrontal cortex during adolescence and addiction

Coordinated activity-induced transcriptional changes across multiple neuron subtypes of the prefrontal cortex (PFC) play a pivotal role in encoding and regulating major cognitive behaviors. Yet, the specific transcriptional programs in each neuron subtype remain unknown. Using single-cell RNA sequencing (scRNA-seq), here we comprehensively classify all unique cell subtypes in the PFC. We analyze transcriptional dynamics of each cell subtype under a naturally adaptive and an induced condition. Adaptive changes during adolescence (between P21 and P60), a highly dynamic phase of postnatal neuroplasticity, profoundly impacted transcription in each neuron subtype, including cell type-specific regulation of genes implicated in major neuropsychiatric disorders. On the other hand, an induced plasticity evoked by chronic cocaine addiction resulted in progressive transcriptional changes in multiple neuron subtypes and became most pronounced upon prolonged drug withdrawal. Our findings lay a foundation for understanding cell type-specific postnatal transcriptional dynamics under normal PFC function and in neuropsychiatric disease states.

Cell-type specific concentration regulation of the basal transcription factor TFIIH in XPBy/y mice model

BackgroundThe basal transcription/repair factor TFIIH is a ten sub-unit complex essential for RNA polymerase II (RNAP2) transcription initiation and DNA repair. In both these processes TFIIH acts as a DNA helix opener, required for promoter escape of RNAP2 in transcription initiation, and to set the stage for strand incision within the nucleotide excision repair (NER) pathway.MethodsWe used a knock-in mouse model that we generated and that endogenously expresses a fluorescent version of XPB (XPB-YFP). Using different microscopy, cellular biology and biochemistry approaches we quantified the steady state levels of this protein in different cells, and cells imbedded in tissues.ResultsHere we demonstrate, via confocal imaging of ex vivo tissues and cells derived from this mouse model, that TFIIH steady state levels are tightly regulated at the single cell level, thus keeping nuclear TFIIH concentrations remarkably constant in a cell type dependent manner. Moreover, we show that individual cellular TFIIH levels are proportional to the speed of mRNA production, hence to a cell’s transcriptional activity, which we can correlate to proliferation status. Importantly, cancer tissue presents a higher TFIIH than normal healthy tissues.ConclusionThis study shows that TFIIH cellular concentration can be used as a bona-fide quantitative marker of transcriptional activity and cellular proliferation.

Nuclei Isolation Staining (NIS) Method for Imaging Chromatin-Associated Proteins in Difficult Cell Types.

Spatial distribution of chromatin-associated proteins provides invaluable information for understanding gene regulation. Conventional immunostaining is widely used for labeling chromatin-associated proteins in many cell types. However, for a subset of difficult cell types, such as differentiated human keratinocytes, achieving high-quality immunostaining for nuclear proteins remains challenging. To overcome this technical barrier, we developed the nuclei isolation staining (NIS) method. In brief, NIS involves rapid isolation of nuclei from live cells, followed by fixation and staining of the nuclei directly on coverslips for subsequent high-magnification imaging. By removing the cytoplasmic contents and staining just the nuclei, this NIS method drastically improves antibody labeling efficiency for chromatin-associated proteins. In this article, we describe the development and a step-by-step protocol of NIS, using differentiated human keratinocytes as an example. We also discuss other applications, based on the principle of this NIS method, for understanding cell-type and cell-state specific gene regulation. © 2019 by John Wiley & Sons, Inc.

2030 - RIBONUCLEASE INHIBITOR (RNH1) REGULATES HEMATOPOIETIC CELL SPECIFIC TRANSLATION

Regulation of gene expression is important for normal development and is mainly controlled at the level of transcription. However, recent studies show that ribosomal proteins (RPs) regulate specific gene expression by selectively facilitating translation of specific mRNAs. Indeed, in Diamond- Blackfan anemia (DBA) and 5q– syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. How mutations in RP genes leads to hematopoietic specific defects is largely unknown. Similar to transcription factors the existence of cell type specific translation regulators remain elusive. Here, we report that Ribonuclease inhibitor (RNH1) regulates hematopoietic cell specific translation. Recently, we published that RNH1 is a ribosomal associated protein and regulates erythropoiesis by regulating GATA1 mRNA translation. In this study, we found that RNH1-deficiency in human hematopoietic origin cells such as erythroid leukemia cells, monocytic cells and T lymphocytes decreased polysome formation but not in non-hematopoietic origin cells such as HEK293, HaCat, HeLa cells. Similarly, OP-Puro incorporation experiments in mice revealed that RNH1-deficency leads to translation defect in hematopoietic cells but not in non- hematopoietic cells. At molecular level, we found that RNH1 binds to ribosomes and regulates RPs gene expression at translation level independent of mTOR signaling. Interestingly, it has been shown that RNH1 expression is translationally down regulated in RPS19 knockdown cells, which is frequently mutated in DBA patients. Supporting RNH1 role in translation, over expression of RNH1 rescues erythroid and translation defects in RPS19 knockdown cells. Collectively, our result unravels the existence of cell type specific translation regulators and may partially explain cell type specific defects caused by mutations in RP genes.

In vivo nuclear capture and molecular profiling identifies Gmeb1 as a transcriptional regulator essential for dopamine neuron function

Midbrain dopamine (mDA) neurons play a central role in reward signaling and are widely implicated in psychiatric and neurodegenerative disorders. To understand how mDA neurons perform these functions, it is important to understand how mDA-specific genes are regulated. However, cellular heterogeneity in the mammalian brain presents a major challenge to obtaining this understanding. To this end, we developed a virus-based approach to label and capture mDA nuclei for transcriptome (RNA-Seq), and low-input chromatin accessibility (liDNase-Seq) profiling, followed by predictive modeling to identify putative transcriptional regulators of mDA neurons. Using this method, we identified Gmeb1, a transcription factor predicted to regulate expression of Th and Dat, genes critical for dopamine synthesis and reuptake, respectively. Gmeb1 knockdown in mDA neurons resulted in downregulation of Th and Dat, as well as in severe motor deficits. This study thus identifies Gmeb1 as a master regulator of mDA gene expression and function, and provides a general method for identifying cell type-specific transcriptional regulators.

MicroRNA-23a contributes to asthma by targeting BCL2 in airway epithelial cells and CXCL12 in fibroblasts.

The deregulated cross-talk between airway epithelial cells with subepithelial fibroblasts during inflammation drives the pathogenesis of asthma. Bioinformatics analysis and luciferase activity assay suggested that B cell lymphoma-2 (BCL2) and CXC ligand 12 (CXCL12) are potential targets of miR-23a. The aim of this study was to elucidate the effect of microRNA-23a (miR-23a) on BCL2, and CXCL12 in asthma. In E3 rats, miR-23a was upregulated in lung tissues after antigen-induced pulmonary inflammation during acute and chronic inflammation. Immunohistochemistry showed downregulation of BCL2 in the epithelium and of CXCL12 in subepithelial fibroblasts and smooth muscle cells. Treatment of isolated cells with miR-23a mimic or inhibitor modified the expression of BCL2 and of CXCL12 in the expected cell type-specific manner. Moreover, in epithelial cells, interleukin-4 upregulated miR-23a expression and thereby decreased the expression of BCL2, while increasing the caspase-3 expression, which was followed by apoptosis. In fibroblasts, the expression of miR-23a was increased by thymic stromal lymphopoietin (TSLP). Consequently, the CXCL12 expression was abrogated. The phosphorylation of CREB was also downregulated by TSLP through the action of miR-23a. This study describes a novel mechanism, where miR-23a is an important cell type-specific regulator for asthma-associated airway wall remodeling parameter. Thus, miR-23a may present a potential new target for the therapy of asthma.

375 The chromatin architectural protein CTCF regulates expression of pro-inflammatory cytokines in the skin cells

Increased evidence suggests that alterations in chromatin structure result in aberrant pro-inflammatory cytokines expression that leads to prolonged inflammatory responses. CCCTC-binding factor (CTCF) is a transcriptional repressor that insulates the expression of neighbouring genes and is involved in chromatin interactions between distal and proximal gene regulatory elements. However, the role of CTCF in the control of pro-inflammatory cytokines expression in the skin cells remains unknown. Here, we show that CTCF is expressed in the nuclei of normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (DF), while its siRNA-mediated deficiency induces cell-type specific changes in pro-inflammatory gene expression. Knockdown of CTCF using siRNAs leads to significant reductions in the transcription of a number of pro-inflammatory genes (TNF, IL1B, IL12B, IL17A, IL19, IL23A, IL36A, CXCL8) in NHEK. In contrast, transcript levels of these genes were markedly up-regulated in CTCF-depleted DF. Furthermore, TNF-alpha treatment causes a more profound inhibitory effect on the CTCF expression in DF compared to NHEK. Finally, we show that CTCF is down-regulated in primary psoriatic fibroblasts compared to DF, while CTCF expression was not affected in psoriatic keratinocytes. Taken together, these data suggest that the chromatin architectural protein CTCF is involved in cell-type specific regulation of pro-inflammatory genes in the skin cells, which suggests its involvement in pathogenesis of inflammatory skin diseases such as psoriasis.

Moving beyond neurons: the role of cell type-specific gene regulation in Parkinson\u2019s disease heritability

Parkinson’s disease (PD), with its characteristic loss of nigrostriatal dopaminergic neurons and deposition of α-synuclein in neurons, is often considered a neuronal disorder. However, in recent years substantial evidence has emerged to implicate glial cell types, such as astrocytes and microglia. In this study, we used stratified LD score regression and expression-weighted cell-type enrichment together with several brain-related and cell-type-specific genomic annotations to connect human genomic PD findings to specific brain cell types. We found that PD heritability attributable to common variation does not enrich in global and regional brain annotations or brain-related cell-type-specific annotations. Likewise, we found no enrichment of PD susceptibility genes in brain-related cell types. In contrast, we demonstrated a significant enrichment of PD heritability in a curated lysosomal gene set highly expressed in astrocytic, microglial, and oligodendrocyte subtypes, and in LoF-intolerant genes, which were found highly expressed in almost all tested cellular subtypes. Our results suggest that PD risk loci do not lie in specific cell types or individual brain regions, but rather in global cellular processes detectable across several cell types.

A new class of constitutively active super-enhancers is associated with fast recovery of 3D chromatin loops

BackgroundSuper-enhancers or stretch enhancers are clusters of active enhancers that often coordinate cell-type specific gene regulation during development and differentiation. In addition, the enrichment of disease-associated single nucleotide polymorphism in super-enhancers indicates their critical function in disease-specific gene regulation. However, little is known about the function of super-enhancers beyond gene regulation.ResultsIn this study, through a comprehensive analysis of super-enhancers in 30 human cell/tissue types, we identified a new class of super-enhancers which are constitutively active across most cell/tissue types. These ‘common’ super-enhancers are associated with universally highly expressed genes in contrast to the canonical definition of super-enhancers that assert cell-type specific gene regulation. In addition, the genome sequence of these super-enhancers is highly conserved by evolution and among humans, advocating their universal function in genome regulation. Integrative analysis of 3D chromatin loops demonstrates that, in comparison to the cell-type specific super-enhancers, the cell-type common super-enhancers present a striking association with rapidly recovering loops.ConclusionsIn this study, we propose that a new class of super-enhancers may play an important role in the early establishment of 3D chromatin structure.

Myeloid TBK1 Signaling Contributes to the Immune Response to Influenza.

Macrophages provide key elements of the host response to influenza A virus (IAV) infection, including expression of type I IFN and inflammatory cytokines and chemokines. TBK1 (TNF receptor-associated factor family member-associated NF-κB activator-binding kinase 1) contributes to IFN expression and antiviral responses in some cell types, but its role in the innate response to IAV in vivo is unknown. We hypothesized that macrophage TBK1 contributes to both IFN and non-IFN components of host defense and IAV pathology. We generated myeloid-conditional TBK1 knockout mice and assessed the in vitro and in vivo consequences of IAV infection. Myeloid-specific loss of TBK1 in vivo resulted in less severe host response to IAV, as assessed by decreased mortality, weight loss, and hypoxia and less inflammatory changes in BAL fluid relative to wild-type mice despite no differences in viral load. Mice lacking myeloid TBK1 showed less recruitment of CD64+SiglecF-Ly6Chi inflammatory macrophages, less expression of inflammatory cytokines in the BAL fluid, and less expression of both IFN regulatory factor and NF-κB target genes in the lung. Analysis of sorted alveolar macrophages, inflammatory macrophages, and lung interstitial macrophages revealed that each subpopulation requires TBK1 for distinct components of the response to IAV infection. Our findings define roles for myeloid TBK1 in IAV-induced lung inflammation apart from IFN type I expression and point to myeloid TBK1 as a central and cell type-specific regulator of virus-induced lung damage.

UV-B response is modulated by cell-type specific signaling pathway in multicellular green algae Volvox carteri

A fundamental question in biology is how multicellular organisms regulate cellular and physiological processes in response to environmental signals in a tissue/cell type-specific manner. Light is one such cue but little is known about its effect on molecular mechanisms underlying cell-type specific signaling. The Volvox genus presents a Germ-Soma differentiation that can be used to understand the genetic mechanisms of evolutionary transition from single-cell to multicellular organisms. Here we report the transcriptional analysis throughout both asexual and sexual life cycles of Volvox carteri in two different cell types under UV-B light irradiation. Our data show that VcUVR8-V1, the main splice variant of the VcUVR8 transcript, accumulates during initiation of cleavage division. Moreover, the transcript level of VcUVR8-V1 increases in response to the sex inducer. VcUVR8 expression seems to remain the same in both cell types, while VcCOP1, the interacting partner of VcUVR8, is expressed in a cell-type specific manner. Interestingly, illumination with low doses of UV-B leads to an increase of VcCOP1 transcript levels only in the somatic cells. Our results indicate that UV-B signaling pathway is differentially regulated between two cell types and environmental UV-B could be involved in cell-type specific regulation of developmental and physiological processes.

Neuron-specific signatures in the chromosomal connectome associated with schizophrenia risk

To explore the developmental reorganization of the three-dimensional genome of the brain in the context of neuropsychiatric disease, we monitored chromosomal conformations in differentiating neural progenitor cells. Neuronal and glial differentiation was associated with widespread developmental remodeling of the chromosomal contact map and included interactions anchored in common variant sequences that confer heritable risk for schizophrenia. We describe cell type-specific chromosomal connectomes composed of schizophrenia risk variants and their distal targets, which altogether show enrichment for genes that regulate neuronal connectivity and chromatin remodeling, and evidence for coordinated transcriptional regulation and proteomic interaction of the participating genes. Developmentally regulated chromosomal conformation changes at schizophrenia-relevant sequences disproportionally occurred in neurons, highlighting the existence of cell type-specific disease risk vulnerabilities in spatial genome organization.

The Differences in Local Translatome across Distinct Neuron Types Is Mediated by Both Baseline Cellular Differences and Post-transcriptional Mechanisms.

Local translation in neurites is a phenomenon that enhances the spatial segregation of proteins and their functions away from the cell body, yet it is unclear how local translation varies across neuronal cell types. Further, it is unclear whether differences in local translation across cell types simply reflect differences in transcription or whether there is also a cell type-specific post-transcriptional regulation of the location and translation of specific mRNAs. Most of the mRNAs discovered as being locally translated have been identified from hippocampal neurons because their laminar organization facilitates neurite-specific dissection and microscopy methods. Given the diversity of neurons across the brain, studies have not yet analyzed how locally translated mRNAs differ across cell types. Here, we used the SynapTRAP method to harvest two broad cell types in the mouse forebrain: GABAergic neurons and layer 5 projection neurons. While some transcripts overlap, the majority of the local translatome is not shared across these cell types. In addition to differences driven by baseline expression levels, some transcripts also exhibit cell type-specific post-transcriptional regulation. Finally, we provide evidence that GABAergic neurons specifically localize mRNAs for peptide neurotransmitters, including somatostatin and cortistatin, suggesting localized production of these key signaling molecules in the neurites of GABAergic neurons. Overall, this work suggests that differences in local translation in neurites across neuronal cell types are poised to contribute substantially to the heterogeneity in neuronal phenotypes.

Abstract P293: Chronic Pressure Overload Impacts Cardiac Endothelial Cell and Macrophage Gene Expression

Introduction: The heart is composed of various different cell types that show distinct differences in gene expression. Inflammation is a key driver of adverse cardiac remodeling and endothelial cells and macrophages are crucially involved in that process. Thus, the aim of this study was to investigate cell type-specific changes in gene expression in cardiac endothelial cells and macrophages after left ventricular pressure overload. Methods and Results: Wildtype mice underwent transverse aortic constriction (TAC) to induce cardiac remodeling. After pressure overload, the number of macrophages in the heart increased about 3-fold (TAC 1755 ± 214 vs. CTRL 533 ± 76 macrophages / mg tissue, P < 0.001) as determined by flow cytometry. Lectin + endothelial cells and CD45 + CD11b + F4/80 high Ly6C low macrophages from CTRL and pressure overloaded hearts were isolated by fluorescence-assisted cell sorting. Cell type-specific gene expression was determined by RNAseq. We found 207 genes differentially expressed in cardiac endothelial cells isolated from pressure overloaded vs. CTRL hearts (n = 3-4 per group, q < 0.05). Approximately 25 % (52 genes) of these genes were highly enriched in endothelial cells vs. heart tissue (> 8-fold, q < 0.05). In cardiac macrophages, 1.359 genes were differentially expressed after pressure overload (n = 4-5 per group, q < 0.05). Bioinformatics analysis were performed to identify affected gene networks and putative key transcription factors involved in the regulation of these genes during cardiac remodeling. Integration of these datasets allowed to determine common, distinct and potentially interacting features of gene expression in both cell types. Conclusion: Chronic pressure overload induces distinct changes in gene expression in isolated cardiac endothelial cells and macrophages. Ongoing epigenetic analysis will provide insight into mechanisms of cell type-specific transcriptional regulation in these cells during cardiac remodeling.

Cell type-specific CLIP reveals that NOVA regulates cytoskeleton interactions in motoneurons

BackgroundAlternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system. This is believed to involve differential regulation of RNA processing in various cell types. However, in vivo study of cell type-specific post-transcriptional regulation has been a challenge. Here, we describe a sensitive and stringent method combining genetics and CLIP (crosslinking and immunoprecipitation) to globally identify regulatory interactions between NOVA and RNA in the mouse spinal cord motoneurons.ResultsWe developed a means of undertaking motoneuron-specific CLIP to explore motoneuron-specific protein–RNA interactions relative to studies of the whole spinal cord in mouse. This allowed us to pinpoint differential RNA regulation specific to motoneurons, revealing a major role for NOVA in regulating cytoskeleton interactions in motoneurons. In particular, NOVA specifically promotes the palmitoylated isoform of the cytoskeleton protein Septin 8 in motoneurons, which enhances dendritic arborization.ConclusionsOur study demonstrates that cell type-specific RNA regulation is important for fine tuning motoneuron physiology and highlights the value of defining RNA processing regulation at single cell type resolution.

Abstract 106: Identifying Cardiac Conduction System-specific Enhancers That Link Cell-type Specification With Human Conduction Defects

The Cardiac Conduction System (CCS) is composed of a small set of specialized cardiomyocytes (CMs) that generate and propagate the electrical impulse required for contraction of the cardiac chambers. Genome Wide Association Studies (GWAS) have identified nearly fifty cardiac rhythm associated Single Nucleotide Polymorphisms (SNPs), and recent reports have characterized how selected SNPs impact CCS gene regulatory networks to influence cardiac conduction. However, a global view of CCS regulatory networks remains to be elucidated, and the vast majority of cardiac conduction associated SNPs remain uncharacterized. Such in-depth analyses have been hampered by our inability to specifically interrogate genome accessibility and RNA expression in the CCS. In order to identify genome-wide cis regulatory elements that correlate with CCS cell-type specification, we generated and rigorously characterized novel knock-in (KI) Cre driver lines that label the Atrio-Ventricular Node (AVN) and ventricular conduction system (VCS) to add to the limited CCS-specific genetic toolkit. In our study, we also used a previously characterized Shox2 KI-Cre driver to label the sinoatrial node (SAN), the primary pacemaker of the heart. Given a variety of issues associated with FACS and Laser Capture Microdissection, we optimized the INTACT (Isolation of Nuclei TAgged in specific Cell Types) method for mammalian heart tissue. To isolate cell-type-specific nuclei, we used Rosa26-Sun1-myc-sfGFP-Tag mice in combination with CCS-specific Cre driver lines. Following isolation and enrichment of CCS-specific nuclei, we performed Omni-ATAC-seq to identify open regions of chromatin (e.g. enhancer elements) and differentially enriched transcription factor motifs for individual CCS cellular sub-types without prior knowledge of cell-type specific regulators. Most cardiac transcriptomes in the literature do not distinguish between individual subtypes. Therefore, using bulk nuclear RNA-Seq, we also generated CCS-subtype specific transcriptomes. We anticipate that our datasets will provide an important starting point for establishing mechanistic links between CCS-specific enhancer elements and disease-associated SNPs identified by GWAS.

Expression of HLA-DQA1 and HLA-DQB1 genes in B lymphocytes, monocytes and whole blood.

Differential expression of HLA-DQA1 and HLA-DQB1 gene alleles was analysed in three different cell populations isolated from peripheral blood-B lymphocytes, monocytes and whole-blood cells. Interallelic differences in mRNA levels were observed: DQA1*03 alleles were among the most expressed in all cell types, whereas DQA1*05 alleles were least expressed in whole blood and monocytes and among the most expressed in B cells. For DQB1 gene, DQB1*06 group of alleles were the most expressed, and DQB1*02 group the least expressed within all cell populations examined. In comparison with the rest alleles, DQB1*06 and DQB1*05:02 alleles have higher expression in monocytes than in B cells, professional antigen-presenting cells. Cell type-specific regulation of expression was observed as well, with higher and more balanced expression of alleles in B lymphocytes compared to monocytes.

Cell type-specific regulation of ciliary transition zone assembly in vertebrates.

Ciliopathies are life‐threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans. By performing quantitative immunofluorescence studies in RPGRIP1L−/− mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type‐specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate‐specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.

Dissection of MAPK signaling specificity through protein engineering in a developmental context

BackgroundMitogen-activated protein kinases (MAPK) signaling affects many processes, some of which have different outcomes in the same cell. In Arabidopsis, activation of a MAPK cascade consisting of YODA, MKK4/5 and MPK3/6 inhibits early stages of stomatal developmental, but the ability to halt stomatal progression is lost at the later stage when guard mother cells (GMCs) transition to guard cells (GCs). Rather than downregulating cascade components, stomatal precursors must have a mechanism to prevent late stage inhibition because the same MKKs and MPKs mediate other physiological responses.ResultsWe artificially activated the MAPK cascade using MKK7, another MKK that can modulate stomatal development, and found that inhibition of stomatal development is still possible in GMCs. This suggests that MKK4/5, but not MKK7, are specifically prevented from inhibiting stomatal development. To identify regions of MKKs responsible for cell-type specific regulation, we used a domain swap approach with MKK7 and a battery of in vitro and in vivo kinase assays. We found that N-terminal regions of MKK5 and MKK7 establish specific signal-to-output connections like they do in other organisms, but they do so in combination with previously undescribed modules in the C-terminus. One of these modules encoding the GMC-specific regulation of MKK5, when swapped with sequences from the equivalent region of MKK7, allows MKK5 to mediate robust inhibition of late stomatal development.ConclusionsBecause MKK structure is conserved across species, the identification of new MKK specificity modules and signaling rules furthers our understanding of how eukaryotes create specificity in complex biological systems.

Epigenetic and Posttranscriptional Regulation of CD16 Expression during Human NK Cell Development.

The surface receptor FcγRIIIA (CD16a) is encoded by the FCGR3A gene and is acquired by human NK cells during maturation. NK cells bind the Fc portion of IgG via CD16a and execute Ab-dependent cell-mediated cytotoxicity, which is critical for the effectiveness of several antitumor mAb therapies. The role of epigenetic regulatory mechanisms controlling transcriptional and posttranscriptional CD16 expression in NK cells is unknown. In this study, we compared specific patterns of DNA methylation and expression of FCGR3A with FCGR3B, which differ in cell type-specific expression despite displaying nearly identical genomic sequences. We identified a sequence within the FCGR3A promoter that selectively exhibits reduced methylation in CD16a+ NK cells versus CD16a- NK cells and neutrophils. This region contained the transcriptional start site of the most highly expressed CD16a isoform in NK cells. Luciferase assays revealed remarkable cell-type specificity and methylation-dependent activity of FCGR3A- versus FCGR3B-derived sequences. Genomic differences between FCGR3A and FCGR3B are enriched at CpG dinucleotides, and mutation of variant CpGs reversed cell-type specificity. We further identified miR-218 as a posttranscriptional negative regulator of CD16a in NK cells. Forced overexpression of miR-218 in NK cells knocked down CD16a mRNA and protein expression. Moreover, miR-218 was highly expressed in CD16a- NK cells compared with CD16a+ NK cells. Taken together, we propose a system of FCGR3A regulation in human NK cells in which CpG dinucleotide sequences and concurrent DNA methylation confer developmental and cell type-specific transcriptional regulation, whereas miR-218 provides an additional layer of posttranscriptional regulation during the maturation process.