m6A RNA modification regulates innate lymphoid cell responses in a lineage-specific manner.

Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune protection. How the post-transcriptional machinery processes different stimuli and initiates robust gene expression in ILCs is poorly understood. Here, we show that deletion of the N6-methyladenosine (m6A) writer protein METTL3 has little impact on ILC homeostasis or cytokine-induced ILC1 or ILC3 responses but significantly diminishes ILC2 proliferation, migration and effector cytokine production and results in impaired antihelminth immunity. m6A RNA modification supports an increase in cell size and transcriptional activity in activated ILC2s but not in ILC1s or ILC3s. Among other transcripts, the gene encoding the transcription factor GATA3 is highly m6A methylated in ILC2s. Targeted m6A demethylation destabilizes nascent Gata3 mRNA and abolishes the upregulation of GATA3 and ILC2 activation. Our study suggests a lineage-specific requirement of m6A for ILC2 responses.

Histone methylation antagonism drives tumor immune evasion in squamous cell carcinomas.

How cancer-associated chromatin abnormalities shape tumor-immune interaction remains incompletely understood. Recent studies have linked DNA hypomethylation and de-repression of retrotransposons to anti-tumor immunity through the induction of interferon response. Here, we report that inactivation of the histone H3K36 methyltransferase NSD1, which is frequently found in squamous cell carcinomas (SCCs) and induces DNA hypomethylation, unexpectedly results in diminished tumor immune infiltration. In syngeneic and genetically engineered mouse models of head and neck SCCs, NSD1-deficient tumors exhibit immune exclusion and reduced interferon response despite high retrotransposon expression. Mechanistically, NSD1 loss results in silencing of innate immunity genes, including the type III interferon receptor IFNLR1, through depletion of H3K36 di-methylation (H3K36me2) and gain of H3K27 tri-methylation (H3K27me3). Inhibition of EZH2 restores immune infiltration and impairs the growth of Nsd1-mutant tumors. Thus, our work uncovers a druggable chromatin cross talk that regulates the viral mimicry response and enables immune evasion of DNA hypomethylated tumors.

A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

Epithelial Wntless regulates postnatal alveologenesis.

Alveologenesis requires the coordinated modulation of the epithelial and mesenchymal compartments to generate mature alveolar saccules for efficient gas exchange. However, the molecular mechanisms underlying the epithelial-mesenchymal interaction during alveologenesis are poorly understood. Here, we report that Wnts produced by epithelial cells are crucial for neonatal alveologenesis. Deletion of the Wnt chaperone protein Wntless homolog (Wls) disrupts alveolar formation, resulting in enlarged saccules in Sftpc-Cre/Nkx2.1-Cre; Wlsloxp/loxp mutants. Although commitment of the alveolar epithelium is unaffected, α-SMA+ mesenchymal cells persist in the alveoli, accompanied by increased collagen deposition, and mutants exhibit exacerbated fibrosis following bleomycin challenge. Notably, α-SMA+ cells include a significant number of endothelial cells resembling endothelial to mesenchymal transition (EndMT), which is also present in Ager-CreER; Wlsloxp/loxp mutants following early postnatal Wls deletion. These findings provide initial evidence that epithelial-derived Wnts are crucial for the differentiation of the surrounding mesenchyme during early postnatal alveologenesis.

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and immunothrombosis.

Severe acute respiratory syndrome coronavirus 2 (SARS­CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS­CoV-2 infections and that CASP4 expression correlates with severity of SARS­CoV-2 infection in humans. SARS­CoV-2­infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS­CoV-2­infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1ß, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS­CoV-2­induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

KLHL21 suppresses gastric tumourigenesis via maintaining STAT3 signalling equilibrium in stomach homoeostasis.

OBJECTIVE: Precancerous metaplasia transition to dysplasia poses a risk for subsequent intestinal-type gastric adenocarcinoma. However, the molecular basis underlying the transformation from metaplastic to cancerous cells remains poorly understood. DESIGN: An integrated analysis of genes associated with metaplasia, dysplasia was conducted, verified and characterised in the gastric tissues of patients by single-cell RNA sequencing and immunostaining. Multiple mouse models, including homozygous conditional knockout Klhl21-floxed mice, were generated to investigate the role of Klhl21 deletion in stemness, DNA damage and tumour formation. Mass-spectrometry-based proteomics and ribosome sequencing were used to elucidate the underlying molecular mechanisms. RESULTS: Kelch-like protein 21 (KLHL21) expression progressively decreased in metaplasia, dysplasia and cancer. Genetic deletion of Klhl21 enhances the rapid proliferation of Mist1+ cells and their descendant cells. Klhl21 loss during metaplasia facilitates the recruitment of damaged cells into the cell cycle via STAT3 signalling. Increased STAT3 activity was confirmed in cancer cells lacking KLHL21, boosting self-renewal and tumourigenicity. Mechanistically, the loss of KLHL21 promotes PIK3CB mRNA translation by stabilising the PABPC1-eIF4G complex, subsequently causing STAT3 activation. Pharmacological STAT3 inhibition by TTI-101 elicited anticancer effects, effectively impeding the transition from metaplasia to dysplasia. In patients with gastric cancer, low levels of KLHL21 had a shorter survival rate and a worse response to adjuvant chemotherapy. CONCLUSIONS: Our findings highlighted that KLHL21 loss triggers STAT3 reactivation through PABPC1-mediated PIK3CB translational activation, and targeting STAT3 can reverse preneoplastic metaplasia in KLHL21-deficient stomachs.

Airway Basal Stem Cells in COVID-19 Exhibit a Proinflammatory Signature and Impaired Mucocililary Differentiation.

Airway basal stem cells (BSCs) play a critical role in epithelial regeneration. Whether coronavirus disease (COVID-19) affects BSC function is unknown. Here, we derived BSC lines from patients with COVID-19 using tracheal aspirates (TAs) to circumvent the biosafety concerns of live-cell derivation. We show that BSCs derived from the TAs of control patients are bona fide bronchial BSCs. TA BSCs from patients with COVID-19 tested negative for severe acute respiratory syndrome coronavirus 2 RNA; however, these so-termed COVID-19-exposed BSCs in vitro resemble a predominant BSC subpopulation uniquely present in patients with COVID-19, manifested by a proinflammatory gene signature and STAT3 hyperactivation. Furthermore, the sustained STAT3 hyperactivation drives goblet cell differentiation of COVID-19-exposed BSCs in an air-liquid interface. Last, these phenotypes of COVID-19-exposed BSCs can be induced in control BSCs by cytokine cocktail pretreatment. Taken together, acute inflammation in COVID-19 exerts a long-term impact on mucociliary differentiation of BSCs.

SOX9 Modulates the Transformation of Gastric Stem Cells Through Biased Symmetric Cell Division.

BACKGROUND & AIMS: Transformation of stem/progenitor cells has been associated with tumorigenesis in multiple tissues, but stem cells in the stomach have been hard to localize. We therefore aimed to use a combination of several markers to better target oncogenes to gastric stem cells and understand their behavior in the initial stages of gastric tumorigenesis. METHODS: Mouse models of gastric metaplasia and cancer by targeting stem/progenitor cells were generated and analyzed with techniques including reanalysis of single-cell RNA sequencing and immunostaining. Gastric cancer cell organoids were genetically manipulated with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for functional studies. Cell division was determined by bromodeoxyuridine-chasing assay and the assessment of the orientation of the mitotic spindles. Gastric tissues from patients were examined by histopathology and immunostaining. RESULTS: Oncogenic insults lead to expansion of SOX9+ progenitor cells in the mouse stomach. Genetic lineage tracing and organoid culture studies show that SOX9+ gastric epithelial cells overlap with SOX2+ progenitors and include stem cells that can self-renew and differentiate to generate all gastric epithelial cells. Moreover, oncogenic targeting of SOX9+SOX2+ cells leads to invasive gastric cancer in our novel mouse model (Sox2-CreERT;Sox9-loxp(66)-rtTA-T2A-Flpo-IRES-loxp(71);Kras(Frt-STOP-Frt-G12D);P53R172H), which combines Cre-loxp and Flippase-Frt genetic recombination systems. Sox9 deletion impedes the expansion of gastric progenitor cells and blocks neoplasia after Kras activation. Although Sox9 is not required for maintaining tissue homeostasis where asymmetric division predominates, loss of Sox9 in the setting of Kras activation leads to reduced symmetric cell division and effectively attenuates the Kras-dependent expansion of stem/progenitor cells. Similarly, Sox9 deletion in gastric cancer organoids reduces symmetric cell division, organoid number, and organoid size. In patients with gastric cancer, high levels of SOX9 are associated with recurrence and poor prognosis. CONCLUSION: SOX9 marks gastric stem cells and modulates biased symmetric cell division, which appears to be required for the malignant transformation of gastric stem cells.

The development and stem cells of the esophagus.

The esophagus is derived from the anterior portion of the foregut endoderm, which also gives rise to the respiratory system. As it develops, the esophageal lining is transformed from a simple columnar epithelium into a stratified squamous cell layer, accompanied by the replacement of unspecified mesenchyme with layers of muscle cells. Studies in animal models have provided significant insights into the roles of various signaling pathways in esophageal development. More recent studies using human pluripotent stem cells (hPSCs) further demonstrate that some of these signaling pathways are conserved in human esophageal development. In addition, a combination of mouse genetics and hPSC differentiation approaches have uncovered new players that control esophageal morphogenesis. In this Review, we summarize these new findings and discuss how the esophagus is established and matures throughout different stages, including its initial specification, respiratory-esophageal separation, epithelial morphogenesis and maintenance. We also discuss esophageal muscular development and enteric nervous system innervation, which are essential for esophageal structure and function.

Helicobacter pylori pathogen inhibits cellular responses to oncogenic stress and apoptosis.

Helicobacter pylori (H. pylori) is a common gastric pathogen that infects approximately half of the world's population. Infection with H. pylori can lead to diverse pathological conditions, including chronic gastritis, peptic ulcer disease, and cancer. The latter is the most severe consequence of H. pylori infection. According to epidemiological studies, gastric infection with H. pylori is the strongest known risk factor for non-cardia gastric cancer (GC), which remains one of the leading causes of cancer-related deaths worldwide. However, it still remains to be poorly understood how host-microbe interactions result in cancer development in the human stomach. Here we focus on the H. pylori bacterial factors that affect the host ubiquitin proteasome system. We investigated E3 ubiquitin ligases SIVA1 and ULF that regulate p14ARF (p19ARF in mice) tumor suppressor. ARF plays a key role in regulation of the oncogenic stress response and is frequently inhibited during GC progression. Expression of ARF, SIVA1 and ULF proteins were investigated in gastroids, H. pylori-infected mice and human gastric tissues. The role of the H. pylori type IV secretion system was assessed using various H. pylori isogenic mutants. Our studies demonstrated that H. pylori infection results in induction of ULF, decrease in SIVA1 protein levels, and subsequent ubiquitination and degradation of p14ARF tumor suppressor. Bacterial CagA protein was found to sequentially bind to SIVA1 and ULF proteins. This process is regulated by CagA protein phosphorylation at the EPIYA motifs. Downregulation of ARF protein leads to inhibition of cellular apoptosis and oncogenic stress response that may promote gastric carcinogenesis.

BMP Signaling in Development, Stem Cells, and Diseases of the Gastrointestinal Tract.

The bone morphogenetic protein (BMP) pathway is essential for the morphogenesis of multiple organs in the digestive system. Abnormal BMP signaling has also been associated with disease initiation and progression in the gastrointestinal (GI) tract and associated organs. Recent studies using animal models, tissue organoids, and human pluripotent stem cells have significantly expanded our understanding of the roles played by BMPs in the development and homeostasis of GI organs. It is clear that BMP signaling regulates GI function and disease progression that involve stem/progenitor cells and inflammation in a tissue-specific manner. In this review we discuss these new findings with a focus on the esophagus, stomach, and intestine.

Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in oesophageal adenocarcinoma.

OBJECTIVE: Oesophageal adenocarcinoma (EAC) arises in the setting of Barrett's oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic bile salts, the mimicry of reflux, in activation of NOTCH signaling in EAC. DESIGN: This study used public databases, EAC cell line models, L2-IL1ß transgenic mouse model and human EAC tissue samples to identify mechanisms of NOTCH activation under reflux conditions. RESULTS: Analysis of public databases demonstrated significant upregulation of NOTCH signaling components in EAC. In vitro studies demonstrated nuclear accumulation of active NOTCH1 cleaved fragment (NOTCH intracellular domain) and upregulation of NOTCH targets in EAC cells in response to reflux conditions. Additional investigations identified DLL1 as the predominant ligand contributing to NOTCH1 activation under reflux conditions. We discovered a novel crosstalk between APE1 redox function, reflux-induced inflammation and DLL1 upregulation where NF-κB can directly bind to and induce the expression of DLL1. The APE1 redox function was crucial for activation of the APE1-NF-κB-NOTCH axis and promoting cancer cell stem-like properties in response to reflux conditions. Overexpression of APE1 and DLL1 was detected in gastro-oesophageal junctions of the L2-IL1ß transgenic mouse model and human EAC tissue microarrays. DLL1 high levels were associated with poor overall survival in patients with EAC. CONCLUSION: These findings underscore a unique mechanism that links redox balance, inflammation and embryonic development (NOTCH) into a common pro-tumorigenic pathway that is intrinsic to EAC cells.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.

Human Airway Basal Cells Undergo Reversible Squamous Differentiation and Reshape Innate Immunity.

Histological and lineage immunofluorescence examination revealed that healthy conducting airways of humans and animals harbor sporadic poorly differentiated epithelial patches mostly in the dorsal noncartilage regions that remarkably manifest squamous differentiation. In vitro analysis demonstrated that this squamous phenotype is not due to intrinsic functional change in underlying airway basal cells. Rather, it is a reversible physiological response to persistent Wnt signaling stimulation during de novo differentiation. Squamous epithelial cells have elevated gene signatures of glucose uptake and cellular glycolysis. Inhibition of glycolysis or a decrease in glucose availability suppresses Wnt-induced squamous epithelial differentiation. Compared with pseudostratified airway epithelial cells, a cascade of mucosal protective functions is impaired in squamous epithelial cells, featuring increased epithelial permeability, spontaneous epithelial unjamming, and enhanced inflammatory responses. Our study raises the possibility that the squamous differentiation naturally occurring in healthy airways identified herein may represent "vulnerable spots" within the airway mucosa that are sensitive to damage and inflammation when confronted by infection or injury. Squamous metaplasia and hyperplasia are hallmarks of many airway diseases, thereby expanding these areas of vulnerability with potential pathological consequences. Thus, investigation of physiological and reversible squamous differentiation from healthy airway basal cells may provide critical knowledge to understand pathogenic squamous remodeling, which is often nonreversible, progressive, and hyperinflammatory.

Transitional basal cells at the squamous-columnar junction generate Barrett's oesophagus.

In several organ systems, the transitional zone between different types of epithelium is a hotspot for pre-neoplastic metaplasia and malignancy, but the cells of origin for these metaplastic epithelia and subsequent malignancies remain unknown. In the case of Barrett's oesophagus, intestinal metaplasia occurs at the gastro-oesophageal junction, where stratified squamous epithelium transitions into simple columnar cells. On the basis of a number of experimental models, several alternative cell types have been proposed as the source of this metaplasia but in all cases the evidence is inconclusive: no model completely mimics Barrett's oesophagus in terms of the presence of intestinal goblet cells. Here we describe a transitional columnar epithelium with distinct basal progenitor cells (p63+KRT5+KRT7+) at the squamous-columnar junction of the upper gastrointestinal tract in a mouse model. We use multiple models and lineage tracing strategies to show that this squamous-columnar junction basal cell population serves as a source of progenitors for the transitional epithelium. On ectopic expression of CDX2, these transitional basal progenitors differentiate into intestinal-like epithelium (including goblet cells) and thereby reproduce Barrett's metaplasia. A similar transitional columnar epithelium is present at the transitional zones of other mouse tissues (including the anorectal junction) as well as in the gastro-oesophageal junction in the human gut. Acid reflux-induced oesophagitis and the multilayered epithelium (believed to be a precursor of Barrett's oesophagus) are both characterized by the expansion of the transitional basal progenitor cells. Our findings reveal a previously unidentified transitional zone in the epithelium of the upper gastrointestinal tract and provide evidence that the p63+KRT5+KRT7+ basal cells in this zone are the cells of origin for multi-layered epithelium and Barrett's oesophagus.

Wnt/Fgf crosstalk is required for the specification of basal cells in the mouse trachea.

Basal progenitor cells are crucial for the establishment and maintenance of the tracheal epithelium. However, it remains unclear how these progenitor cells are specified during foregut development. Here, we found that ablation of the Wnt chaperone protein Gpr177 (also known as Wntless) in mouse tracheal epithelium causes a significant reduction in the number of basal progenitor cells accompanied by cartilage loss in Shh-Cre;Gpr177loxp/loxp mutants. Consistent with the association between cartilage and basal cell development, Nkx2.1+p63+ basal cells are co-present with cartilage nodules in Shh-Cre;Ctnnb1DM/loxp mutants, which maintain partial cell-cell adhesion but not the transcription regulation function of ß-catenin. More importantly, deletion of Ctnnb1 in the mesenchyme leads to the loss of basal cells and cartilage, concomitant with reduced transcript levels of Fgf10 in Dermo1-Cre;Ctnnb1loxp/loxp mutants. Furthermore, deletion of Fgf receptor 2 (Fgfr2) in the epithelium also leads to significantly reduced numbers of basal cells, supporting the importance of Wnt/Fgf crosstalk in early tracheal development.

Use of hPSC-derived 3D organoids and mouse genetics to define the roles of YAP in the development of the esophagus.

Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium.

Targeting SOX2 Protein with Peptide Aptamers for Therapeutic Gains against Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a predominant cancer type in developing countries such as China, where ESCC accounts for approximately 90% of esophageal malignancies. Lacking effective and targeted therapy contributes to the poor 5-year survival rate. Recent studies showed that about 30% of ESCC cases have high levels of SOX2. Herein, we aim to target this transcription factor with aptamer. We established a peptide aptamer library and then performed an unbiased screening to identify several peptide aptamers including P42 that can bind and inhibit SOX2 downstream target genes. We further found that P42 overexpression or incubation with a synthetic peptide 42 inhibited the proliferation, migration, and invasion of ESCC cells. Moreover, peptide 42 treatment inhibited the growth and metastasis of ESCC xenografts in mouse and zebrafish. Further analysis revealed that P42 overexpression led to alternations in the levels of proteins that are important for the proliferation and migration of ESCC cells. Taken together, our study identified the peptide 42 as a key inhibitor of SOX2 function, reducing the proliferation and migration of ESCC cells in vitro and in vivo, and thereby offering a potential therapy against ESCC.

A CRISPR/Cas9 screen identifies the histone demethylase MINA53 as a novel HIV-1 latency-promoting gene (LPG).

Although combination antiretroviral therapy is potent to block active replication of HIV-1 in AIDS patients, HIV-1 persists as transcriptionally inactive proviruses in infected cells. These HIV-1 latent reservoirs remain a major obstacle for clearance of HIV-1. Investigation of host factors regulating HIV-1 latency is critical for developing novel antiretroviral reagents to eliminate HIV-1 latent reservoirs. From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylase, MINA53, is potentially a novel HIV-1 latency-promoting gene (LPG). We next validated MINA53's function in maintenance of HIV-1 latency by depleting MINA53 using the alternative RNAi approach. We further identified that in vitro MINA53 preferentially demethylates the histone substrate, H3K36me3 and that in cells MINA53 depletion by RNAi also increases the local level of H3K36me3 at LTR. The effort to map the downstream effectors unraveled that H3K36me3 has the cross-talk with another epigenetic mark H4K16ac, mediated by KAT8 that recognizes the methylated H3K36 and acetylated H4K16. Removing the MINA53-mediated latency mechanisms could benefit the reversal of post-integrated latent HIV-1 proviruses for purging of reservoir cells. We further demonstrated that a pan jumonji histone demethylase inhibitor, JIB-04, inhibits MINA53-mediated demethylation of H3K36me3, and JIB-04 synergizes with other latency-reversing agents (LRAs) to reactivate latent HIV-1.