Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease resulting from loss-of-function mutations in SEC23 homolog B (SEC23B). We have previously shown that increased expression of SEC23A, a paralogous protein for SEC23B, rescues the CDAII erythroid defect. Here, we generated a human erythroid cell line that expresses enhanced green fluorescent protein (eGFP) from the endogenous SEC23A locus and performed a small-molecule screen to identify compounds that increased SEC23A-eGFP abundance. The top compound passing all filters was an inhibitor of lysine-specific demethylase 1 (LSD1). We found that LSD1 inhibition with RN1 resulted in increased SEC23A expression in erythroid cells derived from human hematopoietic stem and progenitor cells (HSPCs) at doses that did not impair erythroid cell growth or differentiation and rescued the erythroid defect resulting from SEC23B deletion. Genetic down-regulation of LSD1 led to a marked increase in SEC23A mRNA expression in HSPC-derived erythroid cells. Deletion of Lsd1 in mouse erythroid cells resulted in increased Sec23a expression, and RN1 treatment ameliorated the erythroid defect observed in a CDAII mouse model. Mechanistically, we found that LSD1 occupied a sequence in the SEC23A promoter, repressing SEC23A transcription. Deletion of the promotor sequence occupied by LSD1 resulted in increased SEC23A expression and amelioration of CDAII. These findings highlight that LSD1 represses SEC23A transcription and that LSD1 inhibition results in de-repression of SEC23A expression and amelioration of the CDAII erythroid defect, suggesting promising therapeutic strategies for CDAII.

Abstract Background: CBP/p300 are essential transcriptional coactivators of the androgen receptor and play a critical role in prostate cancer pathogenesis. Small-molecule CBP/p300 degraders have demonstrated potent efficacy against prostate cancer but are limited by toxicity, impeding clinical translation. Methods: To enhance tumor specificity and minimize systemic toxicity, we developed a Degrader-Antibody Conjugate (DAC) by linking a highly potent CBP/p300 degrader to a prostate-specific membrane antigen (PSMA) antibody, generating JZY-2233. In vitro antiproliferative activities were assessed using VCaP and LNCaP prostate cancer cell lines. In vivo efficacy, pharmacodynamics, and toxicity were evaluated in a LNCaP xenograft mouse model. Results: JZY-2233 induced robust growth inhibition in VCaP and LNCaP cells, with picomolar IC50 values. In vivo, treatment with JZY-2233 led to sustained CBP/p300 degradation and suppression of c-Myc and PSA in tumor tissues, with effects persisting up to 168 hours post-dose. A single administration at 10 mg/kg resulted in complete tumor suppression for over 90 days in the LNCaP model, significantly exceeding the effect of the unconjugated parent degrader. Importantly, JZY-2233 markedly reduced systemic toxicity. Conclusions: JZY-2233 is a highly potent, antigen-targeted CBP/p300-PSMA DAC, offering prolonged tumor suppression and reduced toxicity in models of advanced prostate cancer. This approach provides a strong rationale for further evaluation in clinical studies and may be adapted for targeting additional tumor antigens across diverse cancers. Citation Format: Mi Wang, Jianzhang Yang, Brandon Bordeau, Shicheng Jin, Yu Wang, Shaomeng Wang. JZY-2233: An antigen-targeted CBP/p300 degrader-antibody conjugate for advanced prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 450.

Analysis of Diamond Blackfan anemia syndrome (DBAS) cohorts and animal models have not revealed a potential mechanism for the variable anemia phenotype, a key feature of this disease. Here, we utilized an established Rpl5Skax23-Jus/+ murine DBAS model in order to study this dynamic erythropoiesis deficiency. These haploinsufficient mice exhibit variably penetrant craniofacial and cardiac defects mimicking the phenotypes of DBAS patients bearing RPL5 mutations. We additionally discovered that this specific heterozygous splicing mutation is pathogenic and leads to partial intron retention. By examining the transcriptome of fetal liver erythroid progenitors at E12.5, we demonstrate downregulation of erythroid differentiation pathways consistent with the DBAS phenotype. We also identified dysregulated transcription of lipid metabolism genes with significant reduction in Scd1 expression in the subset of E12.5 mutant embryos at risk for erythroid failure. SCD1, a key enzyme that converts saturated to monounsaturated fatty acids, has not been previously linked to erythropoiesis or DBAS. When anemia was induced in adult Rpl5Skax23-Jus/+ mice, mutant mice exhibited delayed erythroid recovery, whereas pretreatment with an SCD1 inhibitor resulted in improved erythropoiesis in both wildtype and mutant mice. This analysis suggests a potential role of lipid metabolism in the variable anemia penetrance in DBAS and highlights a previously unappreciated pathway that requires further study as a potential target for drug development.

Loss-of-function mutations in DEPDC5 (DEP domain-containing protein 5), a critical negative regulator of mTORC1 (mechanistic Target of Rapamycin Complex 1), are often identified in patients with refractory epilepsy. To understand its underlying pathogenesis and develop novel therapeutics, we used a highly clinically relevant rat model of DEPDC5-related epilepsy and resected human patient tissues to profile the molecular architecture in the dysplastic cortex. We report here that Slc6a5 (solute carrier family 6 member 5 gene), a marker gene for glycinergic inhibitory neurons, is ectopically overexpressed in mutant excitatory neurons in both experimental animal and human tissues. Using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) in utero electroporation (IUE) to simultaneously knock out Depdc5 and Slc6a5 in forebrain excitatory neurons reduces seizure frequency and duration. These data suggest that SLC6A5 plays an important role in the epileptogenesis of DEPDC5-related epilepsy, although the underlying mechanisms remain unclear.

Selective targeting SMARCA2 by degradation represents a promising new therapeutic strategy for human cancers harboring deficient SMARCA4. Herein we report the discovery of highly potent, selective and oral available SMARCA2 PROTAC degraders, as exemplified by SMD-6346. SMD-6346 achieves DC50 = 3.3 nM and D-max > 90% against SMARCA2 and only modest activity against SMARCA4 (DC50 > 1000 nM, D-max = 46%). SMD-6346 potently and effectively inhibits cell growth in SMARCA4-deficient cancer cell lines and displays minimal cell growth inhibition activity in SMARCA2/4 wild-type cancer cell lines. SMD-6346 attains an excellent pharmacokinetic profile and 61% oral bioavailability in mice. Daily oral administration of SMD-6346 induces robust SMARCA2 depletion in tumor tissues in mice and significantly inhibits tumor growth in the H838 SMARCA4-deficient xenograft model in mice. SMD-6346 is a promising, orally bioavailable SMARCA2 degrader for further optimization for the development of a new therapy for SMARCA4-deficient human cancers.

Analysis of neither Diamond Blackfan anemia syndrome (DBAS) cohorts nor animal models has revealed a potential mechanism for the variable anemia phenotype, a key feature of this disease. Here, we utilized an established Rpl5 Skax23-Jus/+ murine DBAS model in order to study this dynamic erythropoiesis deficiency. These haploinsufficient mice exhibit variably penetrant craniofacial and cardiac defects mimicking the phenotypes of DBAS patients bearing RPL5 mutations. We additionally discovered that this specific heterozygous splicing mutation is pathogenic and leads to partial intron retention. By examining the transcriptome of fetal liver erythroid progenitors at E12.5, we demonstrate that the downregulation of erythroid differentiation pathways is consistent with the DBAS phenotype. We also identified dysregulated transcription of lipid metabolism genes with significant reduction in the abundance of Scd1 in a subset of E12.5 mutant embryos at risk for erythroid failure. SCD1, a key enzyme that converts saturated to monounsaturated fatty acids, has not been previously linked to erythropoiesis or DBAS. When anemia was induced in adult mice, pretreatment with an SCD1 inhibitor resulted in improved erythropoiesis. This analysis suggests a key role of lipid metabolism in the variable anemia penetrance in DBAS and highlights a previously unappreciated pathway that may serve as a potential target for drug development. Key Points:The variable anemia in Rpl5 Skax23-Jus/+ mice is triggered by intrinsic/extrinsic stress Rpl5 haploinsufficient murine and human erythroid progenitors exhibit a lipid metabolism signature with downregulation of Scd1 / SCD .

Proper migration and positioning of migrating pyramidal neurons occur in an "inside-out" pattern and are vital for proper cortico-genesis. The contribution of factors produced by migrating neurons in the morphogenesis of a developing cerebral cortex remains largely unknown. Our previous research revealed that a radial-migrating neuron must navigate to bypass its post-migratory predecessor, which resides at the dorsal border of the developing cortical plate, before reaching its final position. This final stage of radial migration ensures adherence to the "inside-out" pattern at the cellular level. However, whether neurons consistently preserve their sequential order during the prolonged radial migration period remains uncertain. In this study, we found that nascent neurons form queues perpendicular to the cortical plate during radial migration, extending across the upper cortical plate by approximately embryonic day 19. Within each queue, the leading neuron acts as a barrier, restricting the radial migration of the following neuron to preserve their order. We further discovered that the Down syndrome cell adhesion molecule (DSCAM) functions cell-type autonomously in both the leading and following neurons to maintain their sequential order. We find that DSCAM in neurons is necessary to generate a gap between migrating neurons by suppressing N-cadherin-mediated adhesion. Without N-cadherin adhesion, the trailing neuron fails to assemble F-actin at the proximal end of the leading process, preventing its expansion and subsequently hindering nucleokinesis. In Dscam null mutant or Cre-induced knock-out, this gap is not observed, allowing N-cadherin adhesion to persist. Consequently, all migrating neurons keep moving directly to the dorsal border of the cortical plate, and the queue of migrating neurons does not form. This research reveals that DSCAM preserves the sequential order of neurons during radial migration, playing a crucial role in "inside-out" cortico-genesis.

Diamond Blackfan anemia syndrome (DBAS) is a heterogeneous genetic disorder mainly caused by de novo heterozygous ribosomal protein variants, including RPL5. Analysis of DBAS cohorts and animal models has not revealed a potential mechanism for the variable anemia phenotype, which is a key feature of this disease. Treatment-independence occurs in approximately 20% of individuals who previously required steroids or red blood cell transfusions. The underlying mechanism of this phenomenon remains unknown. We previously characterized Rpl5Skax23-Jus/+mice and demonstrated a severe defect in erythropoiesis at E12.5, which led to early embryonic demise in some mutants while others (within the same litter) survived and had complete resolution of anemia by 3 weeks of age. In order to further explore the mechanism leading to this defect, we performed timed matings (Rpl5Skax23-Jus/+ x wildtype (WT)) to obtain E12.5 fetal liver (FL) cells, which were sorted by flow cytometry to obtain CD71+ Ter119- early erythroid progenitor cells. Total RNA was extracted and we performed bulk RNA-seq analysis. We divided mutants into two groups based on liver cellularity with the hypothesis that mutants with very low cellularity (M-low) were the ones with impending erythroid failure and death, while those with close to normal cellularity (M-high) had a higher chance of spontaneous recovery. Analysis of RNA-seq data demonstrated downregulation of erythroid differentiation pathways consistent with the DBAS phenotype. We also identified dysregulation of lipid metabolism genes with significant downregulation of Scd1 in the subset of E12.5 mutant embryos at risk for complete erythroid failure (M-low). SCD1 is a key enzyme found in the endoplasmic reticulum, which catalyzes the conversion of saturated to monounsaturated fatty acids. The role of SCD1 and lipid metabolism in erythropoiesis and in DBA is currently unknown. To test the effect of Scd1 downregulation on erythropoiesis, we pretreated adult mice with a SCD1 inhibitor (SCD1-i) or DMSO daily for 2 weeks then administered phenylhydrazine following pretreatment in order to induce anemia.Mutant mice treated with DMSO showed a significant anemia compared with WT whereas SCD1-i treated mice had no or less significant differences in red blood cell counts. In order to explore the effect of the drug on erythropoiesis, we analyzed hematopoietic stem and progenitor cells by flow cytometry. Mice treated with SCD1-i showed a significant increase in CFU-E and decrease in pre-CFU-E counts in the bone marrow when compared to mice treated with DMSO indicating that downregulation of Scd1 is a compensatory mechanism to improve erythropoiesis in DBA. We propose modulation of lipid metabolism and/or SCD1 as a possible mechanism for the variable anemia penetrance in DBAS and as a novel treatment strategy that warrants further study.

MDM2 is a key negative regulator of the tumor suppressor p53 and an attractive target for cancer therapy. We report the discovery of MD-4251, the first orally efficacious MDM2 degrader developed using PROTAC technology. MD-4251 induces potent and rapid MDM2 degradation in RS4;11 cells (DC50 = 0.2 nM; Dmax = 96% at 2 h), leading to robust p53 activation. It selectively inhibits the growth of acute leukemia cell lines with wild-type p53, with minimal activity in p53 mutant lines. MD-4251 shows excellent oral bioavailability in mice, favorable metabolic stability, and no CYP or hERG liabilities. A single oral dose induces sustained MDM2 depletion and attains complete tumor regression in vivo. These results support MD-4251 as a promising therapeutic candidate for cancers through depletion of MDM2.

ABSTRACT:Small molecules that inhibit LSD1 (lysine-specific demethylase 1, KDM1A) have been shown to induce abundant fetal hemoglobin (HbF) levels in red blood cells both in vitro and in vivo, therefore potentially serving as potent and cost-effective therapeutics to treat the β-globinopathies, sickle cell disease (SCD), and β-thalassemia major (TM). However, most LSD1 inhibitors (LSD1is) that induce HbF in vivo are covalent and irreversible, which leads to adverse effects. In this study, we utilized structure-aided drug design to develop potent new reversible LSD1is, leading to robust γ-globin expression in vitro. Moreover, in a mouse model of SCD, oral administration of these novel inhibitors leads to significant HbF elevation and alleviation of multiple features of disease pathology that are the usual consequences of SCD. In addition, we discovered that combined treatment of an LSD1i with a BRD4 degrader (BD-9136) represses the induction of RUNX1 and PU.1, thereby rescuing the erythroid to myeloid lineage conversion that accompanies LSD1is in hematopoiesis. The data indicate that this new generation of LSD1is can effectively induce HbF levels, reduce SCD pathologies, and are well tolerated by oral administration in SCD mice. We anticipate that the combination of these or related binary compounds offer exciting new therapeutic possibilities for treating SCD and TM.

Immune checkpoint blockade (ICB) has transformed cancer therapy. Immunotherapy efficacy depends on dendritic cell (DC)-mediated tumor antigen presentation, T-cell priming and activation. However, the relationship between the key transcription factors in DCs and ICB efficacy remains unknown. Here, we discover ICB reprogrammed the interplay between the STAT3- and STAT5-transcriptional pathways in DCs, thereby activating T-cell immunity and enabling ICB efficacy. Mechanistically, STAT3 restrained the JAK2- and STAT5-transcriptional pathway, determining the fate of DC function. As STAT3 is often activated in the tumor microenvironment (TME), we designed two types of specific PROTAC degraders of STAT3, SD-36 and SD-2301. STAT3-degraders effectively degraded STAT3 in DCs and reprogramed the DC-transcriptional network toward immunogenicity. Furthermore, STAT3-degrader monotherapy was efficacious in treating advanced tumors and ICB-resistant tumors without toxicity in mice. Thus, the crosstalk between the STAT3- and STAT5-transcriptional pathways determines the DC phenotype in the TME and STAT3 degradation holds promise for cancer immunotherapy. Supported in part by research grants from the Breast Cancer Research Foundation, the U.S., NIH/NCI R01 grants (CA217648, CA123088, CA099985, CA193136, CA152470, and CA244509), and the NIH/NCI through the University of Michigan Rogel Cancer Center Grant (CA46592). J.-X.L. and W.J.L. are supported by the Division of Intramural Research, the National Heart, Lung, and Blood Institute, the NIH. Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

The bromodomain-containing protein BRD9 has emerged as an attractive therapeutic target. In the present study, we successfully identified a number of highly potent BRD9 degraders by using two different cereblon ligands developed in our laboratory. Further optimization led to the discovery of CW-3308 as a potent, selective, and orally bioavailable BRD9 degrader. It displayed degradation potency (DC50) < 10 nM and efficiency (D max) > 90% against BRD9 in the G401 rhabdoid tumor and HS-SY-II synovial sarcoma cell lines and had a high degradation selectivity over BRD7 and BRD4 proteins. CW-3308 achieved 91% of oral bioavailability in mice. A single oral dose efficiently reduced the BRD9 protein by >90% in the synovial sarcoma HS-SY-II xenograft tumor tissue. Oral administration effectively inhibited HS-SY-II xenograft tumor growth in mice. CW-3308 is a promising lead compound for further optimization and extensive evaluation for the treatment of synovial sarcoma, rhabdoid tumor, and other BRD9-dependent human diseases.

ABSTRACT:Nuclear receptor TR4 (NR2C2) was previously shown to bind to the -117 position of the γ-globin gene promoters in vitro, which overlaps the more recently described BCL11 transcription factor A (BCL11A) binding site. The role of TR4 in human γ-globin gene repression has not been extensively characterized in vivo, whereas any relationship between TR4 and BCL11A regulation through the γ-globin promoters is unclear at present. We show here that TR4 and BCL11A competitively bind in vitro to distinct, overlapping sequences, including positions overlapping -117 of the γ-globin promoter. We found that TR4 represses γ-globin transcription and fetal hemoglobin accumulation in vivo in a BCL11A-independent manner. Finally, examination of the chromatin occupancy of TR4 within the β-globin locus, compared with BCL11A, shows that both bind avidly to the locus control region and other sites, but only BCL11A binds to the γ-globin promoters at statistically significant frequency. These data resolve an important discrepancy in the literature and, thus, clarify possible approaches to the treatment of sickle cell disease and β-thalassaemia.

CBP/p300 proteins are key epigenetic regulators and promising targets for the treatment of castration-resistant prostate cancer and other types of human cancers. Herein, we report the discovery and characterization of CBPD-268 as an exceptionally potent, effective, and orally efficacious PROTAC degrader of CBP/p300 proteins. CBPD-268 induces CBP/p300 degradation in three androgen receptor-positive prostate cancer cell lines, with DC50 ≤ 0.03 nM and Dmax > 95%, leading to potent cell growth inhibition. It has an excellent oral bioavailability in mice and rats. Oral administration of CBPD-268 at 0.3-3 mg/kg resulted in profound and persistent CBP/p300 depletion in tumor tissues and achieved strong antitumor activity in the VCaP and 22Rv1 xenograft tumor models in mice, including tumor regression in the VCaP tumor model. CBPD-268 was well tolerated in mice and rats and displayed a therapeutic index of >10. Taking these results together, CBPD-268 is a highly promising CBP/p300 degrader as a potential new cancer therapy.

STAT6 is an attractive therapeutic target for human cancers and other human diseases. Starting from a STAT6 ligand with Ki = 3.5 μM binding affinity, we obtained AK-068 with Ki = 6 nM to STAT6 and at least >85-fold binding selectivity over STAT5. Using AK-068 and cereblon ligands, we discovered AK-1690 as the first, potent and selective PROTAC STAT6 degrader. AK-1690 effectively induces degradation of STAT6 protein in cells with DC50 values of as low as 1 nM while showing minimal effect on other STAT members up to 10 μM. A single dose of AK-1690 effectively depletes STAT6 in mouse tissues. Determination of the first cocrystal structure of STAT6 in complex with AK-1690 provides a structural basis for their interactions. AK-1690 is a powerful tool with which to investigate the roles of STAT6 in human diseases and biological processes and a promising lead compound for further optimization.

TR2 and TR4 (NR2C1 and NR2C2, respectively) are evolutionarily conserved nuclear orphan receptors capable of binding direct repeat sequences in a stage-specific manner. Like other nuclear receptors, TR2 and TR4 possess important roles in transcriptional activation or repression with developmental stage and tissue specificity. TR2 and TR4 bind DNA and possess the ability to complex with available cofactors mediating developmental stage-specific actions in primitive and definitive erythrocytes. In erythropoiesis, TR2 and TR4 are required for erythroid development, maturation, and key erythroid transcription factor regulation. TR2 and TR4 recruit and interact with transcriptional corepressors or coactivators to elicit developmental stage-specific gene regulation during hematopoiesis.

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease resulting from loss-of-function mutations in SEC23B, which encodes a component of coat complex protein II (COPII) vesicles. Mammals express two paralogous genes for SEC23, SEC23A and SEC23B, which encode proteins with ~85% amino acid sequence identity. We have previously shown that the SEC23 paralogs have identical interactomes and that SEC23A overlaps in function with SEC23B. Since the SEC23B/SEC23A expression ratio is disproportionately high in human erythroid cells, we reasoned that the endogenous SEC23A expression level is not sufficiently elevated to compensate for loss of SEC23B in CDAII erythroid cells. Indeed, we demonstrated that increasing SEC23A expression, using CRISPR activation or cDNA expression, completely rescues the SEC23B-null erythroid defect. In this study, we generated a human erythroid cell line that expresses eGFP from the endogenous genomic locus of SEC23A and performed a small molecule screen to identify compounds that increase the SEC23A-eGFP level. The top compound passing all filters was an LSD1 inhibitor. We validated that LSD1 inhibition dramatically increased SEC23A mRNA and protein levels in primary erythroid cells derived from CD34+ human hematopoietic stem and progenitor cells (HSPCs), at doses that do not impair erythroid cell growth or differentiation. We also showed that LSD1 inhibition rescues the erythroid differentiation defect resulting from SEC23B deletion in primary human erythroid cells. We validated LSD1 as the target of RN1 by showing that genetic downregulation of LSD1 also led to a profound induction of SEC23A mRNA levels in HPSC-derived erythroid cells. Subsequently, we validated our findings in vivo. We first demonstrated that deletion of Lsd1 in mouse erythroid cells results in increased Sec23a expression. Subsequently, we generated CDAII mice, with inducible deletion of both Sec23b alleles and haploinsufficiency for Sec23a. We showed that RN1 treatment of CDAII mice rescues the erythroid defect observed in these mice. Finally, using CUT&RUN, we found that LSD1 occupies a sequence in the SEC23A promoter, which when mutated, results in increased SEC23A expression and rescue of CDAII. Taken together, these finding suggest that LSD1 occupies the SEC23A promoter, repressing SEC23A transcription and that in the setting of LSD1 inhibition, SEC23A expression is de-repressed, resulting in CDAII rescue. Therefore, using an unbiased screen, we have identified and validated a novel promising therapeutic strategy for CDAII, using a compound that inhibit LSD1 or a genetic approach based on deleting the SEC23A promoter sequence that is occupied by LSD1.

Elevated levels of fetal hemoglobin (HbF) compensate for reduced normal β-globin chains and ameliorate clinical symptoms for patients with beta-globinopathies, such as sickle cell disease (SCD). The embryonic and fetal globin genes, but not the adult β-globin gene, have direct repeat (DR) elements in their promoters, which have been implicated in γ-globin gene repression (Tanabe et al., 2002). We previously purified a DR element-binding protein complex, which we named DRED, and was the first identified HBG gene repressor (Tanabe et al., 2002). DRED activity is conferred by a complex including LSD1, recruited by the nuclear receptors TR2/TR4 (Cui et al., 2011). BCL11A is a well-characterized transcription factor that independently represses the γ-globin genes through binding to their promoters (Liu et al., 2018). We have shown by co-immunoprecipitation that TR4 interacts with BCL11A in HUDEP2 cells. To understand how TR4 acts to repress the γ-globin gene and the relationship of TR4 to BCL11A in γ-globin repression, we characterized the chromatin occupancy of TR4 and BCL11A within the β-globin locus by CUT&RUN (CNR) in HUDEP2 cells. We established HUDEP2 cells bearing TR4 tagged with 3FLAG+3Myc epitopes (FM-TR4) and in the same cells, BCL11A tagged with 3HA+3Myc (HM-BCL11A) epitopes by genome editing. In addition, another HUDEP2 cell line bearing BCL11A tagged with the 3HA+3Myc (HM-BCL11A) epitope as well a cell line bearing TR4 tagged with the 3FLAG+3Myc epitopes (FM-TR4) were generated. The tagged cell lines not only improved the efficiency of antibody targeting, but also facilitated direct comparison of TR4 vs BCL11A chromatin occupancy by binding of the common Myc epitope in FM-TR4 and HM-BCL11A cell lines. Unbiased motif discovery of TR4 CNR binding of these epitopes in all 3 cell lines yielded the motif of a direct repeat consisting of 2 AGGTCA repeat elements with a 1 nt spacer. The exact match of BCL11A motif with one of the direct repeat elements of TR4 motif suggested that BCL11A and TR4 may be compete for binding to the γ-globin promoter. Overall, TR4 and BCL11A showed a similar pattern of occupancy within the globin locus, although of lower intensity. TR4 presented strong interaction with the locus control regions, among which HS2 and HS3 were the highest, consistent with the model of long-distance interaction through chromosomal looping. However, TR4 was found to be significantly less occupied at the γ-globin promoters than BCL11A. To test whether TR4 and BCL11A can bind in vitro to the motifs found in the γ-globin promoters, we performed TR4 and BCL11A electrophoretic mobility shift assays (EMSAs). We showed that TR4 binds to the DR1 element within the γ-globin promoters, which contains the distal TGACCA motif that was bound by BCL11A. Notably, mutation of the TGACCA motif disrupted both BCL11A and TR4 binding, while the other direct motif is indispensable for TR4 binding, supporting overlapping binding of BCL11A and TR4. Interestingly, DNA-TR4 protein complexes were competitively depleted by high concentrations of BCL11A and vice versa, suggesting competitive binding between the two factors. Studies of this mechanism in the erythroid cells are underway. In addition, unbiased motif discovery of TR4 CNR also enriched for KLF1, LRF and NF-Y motifs. Co-IP assays demonstrated interaction between TR4 and KLF1 and LRF, suggesting the presence of a large repressor complex. We also explore genome-wide occupancy of TR4 and discovered strong TR4 interactions at promoters of multiple HbF repressing transcription factors and corepressors, including NFIA and EHMT1, suggesting that the role of TR4 in HbF repression may be partially mediated by regulating expression of various HbF repressor genes. In summary, these data suggest that the function of TR4 in HbF silencing is mediated in part by direct repression of HBG promoters in a competitive manner with BCL11A, and in part by regulating the expression of multiple HbF repressor genes.