It is well established that high levels of fetal hemoglobin (HbF) can overcome the pathophysiologic defects in sickle cell disease (SCD) and beta-thalassemia. Despite the recent success of gene therapy in increasing HbF in SCD and the usefulness of hydroxyurea in improving patient outcomes, there remains a pressing need for more effective small molecules to treat the vast majority of SCD patients who will not benefit from gene therapy in the foreseeable future. In addition to the strong negative regulatory transcription factors BCL11A (Sankaran et al, 2008) and ZBTB7A(LRF) (Masuda et al, 2016), the MBD2 NuRD complex, which recognizes and binds at sites of CpG methylation, is essential for HBG silencing in adult erythroid cells (Rupon et al, 2006; Yu et al, 2019; Shang et al, 2023). It has been shown previously that depletion of CHD4, a critical component of MBD2-NuRD which binds to the core complex through GATAD2A (Gnanapragasam et al, 2011), by as little as 50% induces high levels of HbF without blocking differentiation in primary adult human erythroid cells (Amaya et al, 2013; Lan et al, 2021; Vinjumar et al, 2021). To identify potential targets for small-molecule disruption of MBD2-NuRD, we employed the AlphaFold3 ML program to predict the structure of the protein-protein interaction between GATAD2A and CHD4 that tethers the latter to the MBD2-NuRD complex. This analysis indicated that a small helical region in the CR-2 domain of GATAD2A binds to the interface between the C-terminal domains 1 and 2 (CTD1/2) of CHD4. Intracellular NanoBRET assays confirmed strong binding between the entire CR-2 region or the small helix from this region and CHD4(CTD12) domains. Isothermal calorimetry likewise demonstrated tight binding between these domains in vitro. Furthermore, mutating amino acids in the CR-2 helix, which are predicted to be critical for the interaction, disrupted high-affinity binding to CHD4 (CTD1/2). Enforced expression of wild-type GATAD2A, but not GATAD2A mutated at these critical amino acids, resulted in silencing of HBG expression in HUDEP-2 cells in which GATAD2A was knocked out. Prime editing (PE) of the same critical amino acids in the endogenous GATAD2A(CR-2) region resulted in over 40% HBG/(HBG+HBB) RNA levels and likewise over 40 % HbF levels. Immunoprecipitation assays showed dissociation of CHD4 from GATAD2A in the edited cells. NOME SEQ assays demonstrated the same loss of nucleosomes over the HBG promoter as seen in MBD2 knockout cells, and chromatin immunoprecipitation assays showed markedly decreased occupancy of CHD4. RNA seq analysis was carried out to identify other genes affected by the PE editing. HBG1, HBG2, and BGLT3 were upregulated, and HBB was mildly downregulated, while neither CD235 nor CD71, markers of erythroid differentiation, were affected. Lentiviral vector-mediated expression of the small wild-type CR-2 peptide characterized in binding assays resulted in over 15% HBG/(HBG+HBB) RNA levels and dissociation of GATAD2A from CHD4, while in contrast expression of the corresponding peptide with mutations of critical amino acids at the interface with CHD4 (CTD1/2) did not affect either. Together, these experiments demonstrate an on-target effect of disrupting the GATAD2A interaction with CHD4 to relieve MBD2-NuRD mediated silencing of HBG expression. In summary, structural and biophysical studies guided by AlphaFold3 predictions and followed by functional genetic, biochemical, and cellular assays showed that a small peptide can disrupt the interaction between GATAD2A and CHD4 in the MBD2-NuRD complex, thereby inducing high levels of Hb F in adult human erythroid cells. Given the viability and mild phenotype of MBD2 knockout mice and the clinical success of multiple peptidomimetic drugs, these results identify a specific target for the development of small molecules to therapeutically induce high levels of Hb F in patients with SCD and beta-thalassemia.

Methyl-CpG binding domain 2 (MBD2) is a critical epigenetic regulator that recognizes and binds with high selectivity to methylated CpG dinucleotides, a fundamental epigenetic mark involved in gene regulation and chromatin organization. Therefore, understanding the interactions and conformational dynamics that drive MBD2's high selectivity and strong binding affinity to methylated CpG is crucial to unraveling its regulatory mechanisms. In this study, through extensive classical MD simulations, we investigated the formation of the methylated CpG-MBD2 recognition complex. By positioning MBD2 one base pair downstream of its target mCpG site, we observed its transition to a stable complex at the target site within the microsecond timescale. Significantly, we observe that upon binding to methylated CpG, MBD2 forms two distinct stable complexes, with one state adopting a conformation that agrees well with the X-ray structure of the complex (primary state) and the other exhibiting a distinct binding conformation with lower affinity for methylated CpG (secondary state). Our data indicate that S189 serves as a key macro-switch, where loss of its interaction with the methylcytosine backbone shifts the complex toward the secondary state. This is supported by MD simulations of the S189A mutation, where we observe that the complex adopts a conformation closer to the secondary state. Ultimately, our NMR experiments confirm that the S189A mutation does not alter MBD2's selectivity for methylated CpG, while fluorescence polarization demonstrates a reduction in binding affinity, consistent with our MD simulations. Together, these findings strongly suggest that MBD2 adopts a bistable equilibrium in binding to methylated CpG dinucleotides.

Over the past 50 years, research has uncovered the co-regulatory proteins and complexes that silence the expression of the γ-globin gene in a developmental stage-specific manner. Recent research expanded the list of these regulatory factors by showing that the eight twenty-one protein 2 (ETO2) helps recruit the nucleosome remodeling and deacetylase (NuRD) complex to the globin locus. Furthermore, ETO2 regulates hematopoietic differentiation and is a potential therapeutic target for acute leukemia. In this work, we identify critical interactions between ETO2 and the GATA Zn finger domain containing the 2A (GATAD2A) component of NuRD. The ETO2 nervy homology region 4 (NHR4) domain interacts with multiple polyproline-leucine motifs within GATAD2A. We demonstrate that oligomerization of the ETO2 nervy homology region 3 (NHR3) enhances its affinity for peptides containing at least two polyproline-leucine motifs. Replacing the native motifs from GATAD2A with a higher-affinity sequence from known-binder N-CoR markedly enhances binding affinity, yielding a peptide that disrupts the interaction between ETO2 and target proteins. Enforced peptide expression elevates γ-globin expression levels and induces differentiation of HUDEP-2 and K562 cells. These findings provide insight into ETO2-mediated recruitment of co-regulatory proteins and yield a novel approach for ETO2 inhibition through multivalent binding of the NHR4 domain.

There is increasing evidence that the methyl-binding domain (MBD) is a protein-protein interaction motif that can function independently of methylated DNA binding. The MBD proteins found throughout plants and invertebrates duplicated into multiple vertebrate DNA and non-DNA-binding members (MBD1, MBD2, MBD3, MBD4, MBD5, MBD6, MECP2, BAZ2A, BAZ2B, SETDB1, and SETDB2). Although many invertebrate species possess MBD proteins that can bind and recognize DNA methylation, the DNA-binding function has been independently lost multiple times, with only minor alterations to the protein interaction residues. The nucleosome remodeling and deacetylase (NuRD) complex, which interacts with MBD2/3 and is colocalized with MBD1/4 ChIP-Seq, is maintained in species where MBD2/3 cannot bind to DNA. NuRD ChIP-seq data from HepG2 cell line, human induced pluripotent stem cells (iPSCs), and human iPSC-derived liver cells suggest that the NuRD complex is highly localized to nonmethylated CpG-rich housekeeping gene promoter elements, which are essential in organogenesis and maintained within the Drosophila melanogaster MBD2/3 non-DNA-binding system. Integration of MBD interaction proteins and NuRD gene expression from >115 million cells of single-cell RNA-seq, along with thousands of bulk tissue profiles, highlights a critical role of MBD3, MECP2, and GATAD2B in brain development and intellectual disability syndromes that is maintained throughout invertebrate neural development and likely involves evolutionary expanded entanglement as the vertebrate MBD proteins expanded. This work suggests that MBD has a largely unexplored role as a critical protein interaction motif that is evolutionarily conserved for regulating enhancers and promoters.NEW & NOTEWORTHY The evolution of the methyl-binding domain (MBD) suggests a shared function in gene regulation, from plants to humans, with the conservation of non-DNA-interacting amino acids critical for protein-protein interactions. The MBD-regulated NuRD complex localizes to promoters of housekeeping genes with hypomethylated CpG islands. Expression profiles suggest a shared role for NuRD complex components in neurodevelopment, where the MBD3 and GATAD2B subcomplex of NuRD may be underexplored due to its non-DNA-binding biology.

89 Background: Oxaliplatin hypersensitivity is not uncommon in patients with gastrointestinal (GI) cancers receiving chemotherapy. The use of oral premeditations (dexamethasone, cetirizine and famotidine) starting 24 hours before treatment and continuing for a total of three days, further intravenous premedication (dexamethasone, chlorphenamine and famotidine) 30 minutes before treatment and the gradual administration of oxaliplatin over 6.5 hours in four separate escalating doses (desensitisation protocol) is commonly used in such cases. While desensitisation is an option, especially in severe cases, responses and survival outcomes upon rechallenge are not well described. Methods: A retrospective chart review of patients with various GI malignancies who received oxaliplatin-based desensitisation chemotherapy after a severe drug reaction between October 2019 and October 2022 at a single cancer centre was performed. Clinicopathological characteristics and oncological outcomes were assessed. Results: Forty-four patients with a median age of 61 years (range 38-81) were studied. The majority had a diagnosis of CRC (n=22; 50%), followed by oesophago-gastric (n=19; 4%), appendiceal (n=1), cholangiocarcinoma (n=1), and cancer of unknown primary (n=1). More than two thirds of patients (n=32, 73%) were treated with palliative intent. The most common regimens associated with oxaliplatin hypersensitivity reactions after a median of 3 cycles (range 1-10) were FOLFOX (n=24, 55%), CAPOX (n=18, 41%), FLOT (n=1) and FOLFOXIRI (n=1). Seven patients (16%) had another reaction after desensitisation, leading to discontinuation of treatment. Thirty-seven patients (84%) completed treatment as planned. Treatment outcomes after desensitisation included 5 (11%) patients with no evidence of disease (NED) after adjuvant treatment, 16 (37%) patients with disease control, including 3 (7%) partial responders (PR) and 13 (30%) patients with stable disease (SD), and 23 (52%) patients with progressive disease (PD). Conclusions: Oxaliplatin desensitisation is feasible with low discontinuation rates and leads to acceptable oncological outcomes. All patients should be offered desensitisation to allow continuation of active systemic therapy.

Sufficiently high levels of HbF have been shown to ameliorate the pathophysiologic effects of HbS in sickle cell anemia patients bearing the homozygous codon 6 GAG to GTG mutation in the beta globin gene (HBB).Leukemia Related Factor (LRF, also known as ZBTB7A), BCL11A, and the MBD2-NuRD complex are major mediators of HbF silencing in adult erythroid cells, and combined depletion of both LRF and BCL11A was shown to result in nearly 100% HbF in adult erythroid cells (Masuda, etal, Science 2016). Both LRF and BCL11A are associated with a NuRD complex. In the case of BCL11A we have shown that this is specifically MBD2a-NuRD, and that silencing results from positioning of a nucleosome over the proximal gamma globin gene (HBG) promoter and is at least partially dependent on promoter CpG methylation. (Shang etal, Proc.Natl.Acad.Sci. USA, 2023). To define the specific NuRD complex that participates in LRF mediated HBG silencing we have investigated the roles of both MBD2-NuRD and MBD3-NuRD.NOME SEQ assays performed in MBD2 KO HUDEP-2 cells, showed the loss of a nucleosome positioned over the -189 GATA-1 site binding, which has been shown to be critical for HBG expression, and at which GATA-1 binds when the -198 HBG promoter binding site for LRF is mutated ( Doerfler, etal, Nature Genet., 2021). This suggests that MBD2-NuRD associates with LRF to silence HBG expression, consistent with the lack of effect of MBD3 depletion on HBG expression. When both MBD2 and MBD3 were knocked out in HUDEP-2 cells, over 90% HBG/ (HBG+HBB) RNA levels were observed, similar to the levels observed with knockout of both LRF and BCL11A. However, the increase in HBG/(HBG+HBB) RNA ratio and corresponding HbF/ (HbF+HbA) ratio were due primarily to a 4-5-fold decrease in HBB RNA and protein, as the level of HBG RNA was the same or slightly less than in MBD2 KO cells.Chromatin conformation capture (3C) assays showed that combined depletion of MBD2 and MBD3 resulted in significantly decreased interaction of the Locus Control Region (LCR) with the HBB gene, increased LCR interaction with the BGLT3 locus and a very large increase in interaction of the 3' beta globin locus enhancer with the HBB gene. These results demonstrate that MBD3-NuRD plays a critical role in the interaction of the LCR with the HBB gene to maintain its high level of expression in adult erythroid cells. Depletion of LRF in MBD2 KO HUDEP-2 cells resulted in the same >90% HBG/(HBG+HBB) RNA level, as well as the same decrease in HBB RNA as depletion of MBD3. LRF depletion alone in WT HUDEP-2 cells also resulted in a significant decrease in HBB expression without a significant increase in HBG expression. This contrasts with the effect of mutation of the -198 LRF binding site in the HBG promoter, which results in high levels of HBG expression without a significant decrease in HBB expression ( Antoniou, etal, Nature Comm.,2022). Immunoprecipitation assays showed that LRF interacts with either MBD2-NuRD or MBD3-NuRD. These results are consistent with the fact that the repressive action of LRF, like that of BCL11A, requires association specifically with MBD2-NuRD to position a nucleosome and block the binding of a positive acting transcription factor, in this case GATA-1. Accordingly, disruption of MBD2-NuRD results in high level HBG expression while disruption of MBD3-NuRD does not, as we and others have shown. Conversely, LRF in association with MBD3-NuRD is required for maximum expression of the HBB gene in adult erythroid HUDEP-2 cells. Since combined disruption of the BCL11A-MBD2-NuRD complex and the LRF-MBD3-NuRD complex results in nearly 100% Hb F levels, these findings have implications for the treatment of sickle cell anemia, in which maximizing HbF levels while lowering HbS levels should provide the optimum protection from RBC sickling and its pathophysiologic consequences.

Significance Reversal of fetal hemoglobin (HbF) silencing can ameliorate the effects of sickle cell anemia. Despite available gene therapy and stem cell transplantation modalities, the majority of affected patients worldwide will not have access to these in the near future. Thus, there is a need for safe and effective small-molecule therapeutics. We report here that stable occupancy of a major HbF silencing complex containing BCL11A, MBD2a–NURD, and PRMT5 and exclusion of the transcriptional activator NF-Y at the γ-globin gene promoter require specific features of MBD2a. These results provide a unified model for the relationships between the previously reported HbF silencers MBD2–NuRD, BCL11A, DNA methylation, and PRMT5 that may facilitate development of therapeutic agents to reverse HbF silencing.

Amide−π interactions, in which an amide interacts with an aromatic group, are ubiquitous in biology, yet remain understudied relative to other noncovalent interactions. Recently, we demonstrated that an electrostatically tunable amide−π interaction is key to recognition of histone acyllysine by the AF9 YEATS domain, a reader protein which has emerged as a therapeutic target due to its dysregulation in cancer. Amide isosteres are commonly employed in drug discovery, often to prevent degradation by proteases, and have proven valuable in achieving selectivity when targeting epigenetic proteins. However, like amide−π interactions, interactions of amide isosteres with aromatic rings have not been thoroughly studied despite widespread use. Herein, we evaluate the recognition of a series of amide isosteres by the AF9 YEATS domain using genetic code expansion to evaluate the amide isostere−π interaction. We show that compared to the amide−π interaction with the native ligand, each isostere exhibits similar electrostatic tunability with an aromatic residue in the binding pocket, demonstrating that the isosteres maintain similar interactions with the aromatic residue. We identify a urea‐containing ligand that binds with enhanced affinity for the AF9 YEATS domain, offering a promising starting point for inhibitor development. Furthermore, we demonstrate that carbamate and urea isosteres of crotonyllysine are resistant to enzymatic removal by SIRT1, a protein that cleaves acyl post‐translational modifications, further indicating the potential of amide isosteres in YEATS domain inhibitor development. These results also provide experimental precedent for interactions of these common drug discovery moieties with aromatic rings that can inform computational methods.

The nucleosome remodeling and deacetylase (NuRD) complex modifies nucleosome positioning and chromatin compaction to regulate gene expression. The methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) play a critical role in complex formation; however, the molecular details of how they interact with other NuRD components have yet to be fully elucidated. We previously showed that an intrinsically disordered region (IDR) of MBD2 is necessary and sufficient to bind to the histone deacetylase core of NuRD. Building on that work, we have measured the inherent structural propensity of the MBD2-IDR using solvent and site-specific paramagnetic relaxation enhancement measurements. We then used the AlphaFold2 machine learning software to generate a model of the complex between MBD2 and the histone deacetylase core of NuRD. This model is remarkably consistent with our previous studies, including the current paramagnetic relaxation enhancement data. The latter suggests that the free MBD2-IDR samples conformations similar to the bound structure. We tested this model of the complex extensively by mutating key contact residues and measuring binding using an intracellular bioluminescent resonance energy transfer assay. Furthermore, we identified protein contacts that, when mutated, disrupted gene silencing by NuRD in a cell model of fetal hemoglobin regulation. Hence, this work provides insights into the formation of NuRD and highlights critical binding pockets that may be targeted to block gene silencing for therapy. Importantly, we show that AlphaFold2 can generate a credible model of a large complex that involves an IDR that folds upon binding.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

Sperm-associated antigen 6 (SPAG6) is the mammalian orthologue of Chlamydomonas PF16, an axonemal central pair protein involved in flagellar motility. In mice, two Spag6 genes have been identified. The ancestral gene, on mouse chromosome 2, is named Spag6. A related gene originally called Spag6, localized on mouse chromosome 16, evolved from the ancient Spag6 gene. It has been renamed Spag6-like (Spag6l). Spag6 encodes a 1.6 kb transcript consisting of 11 exons, while Spag6l encodes a 2.4 kb transcript which contains an additional non-coding exon in the 3′-end as well as the 11 exons found in Spag6. The two Spag6 genes share high similarities in their nucleotide and amino acid sequences. Unlike Spag6l mRNA, which is widely expressed, Spag6 mRNA expression is limited to a smaller number of tissues, including the testis and brain. In transfected mammalian cells, SPAG6/GFP is localized on microtubules, a similar localization as SPAG6L. A global Spag6l knockout mouse model was generated previously. In addition to a role in modulating the ciliary beat, SPAG6L has many unexpected functions, including roles in the regulation of ciliogenesis/spermatogenesis, hearing, and the immunological synapse, among others. To investigate the role of the ancient Spag6 gene, we phenotyped global Spag6 knockout mice. All homozygous mutant mice were grossly normal, and fertility was not affected in both males and females. The homozygous males had normal sperm parameters, including sperm number, motility, and morphology. Examination of testis histology revealed normal spermatogenesis. Testicular protein expression levels of selected SPAG6L binding partners, including SPAG16L, were not changed in the Spag6 knockout mice, even though the SPAG16L level was significantly reduced in the Spag6l knockout mice. Structural analysis of the two SPAG6 proteins shows that both adopt very similar folds, with differences in a few amino acids, many of which are solvent-exposed. These differences endow the two proteins with different functional characteristics, even though both have eight armadillo repeats that mediate protein–protein interaction. Our studies suggest that SPAG6 and SPAG6L have different functions in vivo, with the evolved SPAG6L protein being more important. Since the two proteins have some overlapping binding partners, SPAG6 could have functions that are yet to be identified.

Increasing the expression of fetal hemoglobin ameliorates the symptoms of sickle cell disease and β-thalassemia. Therefore, blocking fetal hemoglobin silencing in adult erythrocytes represents a potent strategy for treating these diseases. Extensive research over the past five decades have uncovered critical transcription factors involved in silencing fetal hemoglobin, including Bcl11A and Zbtb7A (LRF), among others. The Nucleosome Remodeling and Deacetylase (NuRD) complex represents a central co-regulatory factor recruited by these proteins and DNA methylation to silence gene expression. Hence, NuRD represents a potential target for blocking silencing by multiple factors simultaneously, thereby leading to robust induction of fetal hemoglobin. We previously have shown that disrupting the MBD2-NuRD complex induces high levels of fetal hemoglobin in both the HUDEP-2 tissue culture cell line and primary patient-derived bone marrow stem cells [Yu et al. (2019) Haematologica, 104(12), 2361-2371]. Recent work by the Dean lab [Guo et al. (2020). Nucleic Acids Research, 48(18), 10226-10240] added to the list of factors that bind and recruit NuRD to the globin locus. They showed that ETO2, a well-established factor in erythroid differentiation, binds directly to NuRD, which required the tetramerization domain of ETO2. However, the specific contacts driving this interaction remain unknown. Based on previous work by the Bushweller [Liu et al. (2007) Cancer Cell, 11(6), 483-497] and Vermeulen [Spruijt et al. (2016) Cell Reports, 17(3), 783-798] groups, we hypothesized that ETO2 binds directly to the GATAD2A component of NuRD through the Nervy Homology Region-4 (NHR4) domain of ETO2 and multiple polyproline-leucine (PPPL) motifs in GATAD2A. Here we use NMR analyses to show that the ETO2-NHR4 domain binds each of four polyproline-leucine motifs in GATAD2A. We measure binding by chemical shift analysis which reveals a weak affinity (KD ~200-300 μM) for the individual peptides. We then show that the ETO2-NHR3 domain contributes to weak homo-oligomerization (dimer-tetramer formation) by gel filtration analysis. Importantly, multivalent interaction between the PPPLx4 region of GATAD2A and a construct including both the NHR3 and NHR4 domains of ETO2 leads to a dramatic increase in binding affinity (KD ~ 4 μM) as measured by isothermal titration calorimetry. Furthermore, using an in-cell bioluminescent resonance energy transfer assay (NanoBRET), we show that including the NHR2 tetramerization domain augments association between these proteins. Mutating key residues in either GATAD2A-PPPLx4 or ETO2-NHR4 domains disrupts this association in cells. Finally, we show that enforced expression of a peptide from GATAD2A containing the four polyproline-leucine motifs consistently induces a low-level of fetal hemoglobin mRNA expression in K-562 cells. Together, these findings establish that the ETO2-NHR4 domain plays a central role in recruitment of NuRD for gene silencing. Tetramerization of the NHR2 and NHR3 domains leads to multivalent association with GATAD2A, which dramatically increases binding affinity. This result has mechanistic implications for fetal hemoglobin silencing and erythroid differentiation, potentially leading to a novel therapeutic target for treating β-hemoglobinopathies. Furthermore, gene rearrangements involving the ETO family of proteins drive the development of acute myeloid leukemia. Our studies provide an explanation for how the stoichiometry of the fusion proteins may dictate which co-regulatory complexes are brought to a specific site and suggest a strategy for selectively targeting these complexes.

The methyl-CpG-binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status.

Axonemal dynein light intermediate polypeptide 1 (DNALI1) was originally cloned from Chlamydomonas reinhardtii in an effort to find motor proteins essential for flagellar motility. Here we report that DNALI1 is a binding partner of parkin co-regulated gene 1 (PACRG), which forms a complex with meiosis expressed gene 1 (MEIG1) in the manchette, a transient and unique structure only present in the elongating spermatids and required for normal spermiogenesis of the male germ cell differentiation process. DNALI1 recruits the PACRG protein in transfected CHO cells, and also stabilizes PACRG in bacteria and transfected mammalian cells. The untagged DNALI1 could also be co-purified with His-tagged PACRG in the gel filtration assay. Immunofluorescence staining on isolated male germ cells revealed that DNALI1 was present in the manchette of elongating spermatids, and colocalized with PACRG in this structure. In Pacrg mutant mice, localization of DNALI1 in the manchette was not changed, suggesting that DNALI1 and PACRG form a complex in the manchette, with DNALI1 being an upstream molecule. Mice deficiency in DNALI1 specifically in male germ cells showed dramatically reduced sperm numbers and were infertile. In addition, majority of the sperm exhibited abnormal morphology including misshapen heads, bent tails and enlarged midpiece, discontinuous accessory structure, and loss of sperm individualization, emphasizing the importance of DNALI1 in sperm development. Examination of testis histology revealed impaired spermiogenesis in the conditional Dnali1 knockout mice. Electron microscopy revealed disrupted ultrastructure in sperm of the Dnali1 mutant mice. Testicular levels of MEIG1, PACRG and SPAG16L proteins were not changed in the Dnali1 mutant mice. However, MEIG1 and SPAG16L were no longer present in the manchette in the absence of DNALI1. These findings demonstrate that DNALI1 is involved in the connection of the MEIG1/PACRG complex to carry cargo proteins along the manchette microtubules for sperm flagella formation. Given that Dnali1 mutant mice showed impaired sperm individualization that was not observed in the MEIG1 nor PACRG-deficient mice, DNALI1 might fulfill other functions beyond its role associated with the MEIG1/PACRG complex. Thus, DNALI1 plays multiple roles in sperm cell differentiation and function. Summary statement Axonemal dynein light intermediate polypeptide 1 (DNALI1) is required for sperm formation and male fertility. It associates with the MEIG1/PACRG complex in the manchette and is involved in a cargo transport system. In addition, it might be related to IFT and sperm individualization.

Abstract Acute myeloid and T-cell acute lymphoblastic leukemia are aggressive subtypes of acute leukemias with 5-year survival rates ranging from 10% to 50%. CHD4, the ATPase chromatin re-modeling component of the NuRD co-repressor complex, has been shown to promote survival of some solid tumor cells (colon cancer, glioblastoma). Our laboratory has shown that CHD4 depletion increases sensitivity of both AML cell lines and primary cells to standard chemotherapy agents, and reduces colony formation in soft agar. Importantly, CD34+ progenitor cells are spared these phenotypic responses. Apart from CHD4, the classical NuRD complex contains either MBD2 or MBD3, and at least four other proteins (GATAD2A/B, HDAC1/2, MTA1/2/3, RBBP4/7). CHD4 is linked to MBD2 or MBD3 via GATAD2A/B proteins through a coiled-coil interaction. Therefore, we sought to determine whether the observed CHD4 effects on AML cell survival are through NuRD, or by acting outside of NuRD. We showed that depletion of either MBD2 or MBD3 did not reproduce the phenotypes observed with CHD4 depletion in U937 (AML) cell lines. However, depletion of both MBD2 and MBD3 resulted in a four-fold reduction in colony numbers in soft agar, and a two to three fold increased sensitivity to cytosine arabinoside (AraC) and daunorubicin (DNR) (AACR, 2019). In order to determine if these findings hold true for other poor prognosis acute leukemias, we have knocked down CHD4 in T-ALL cell lines (Jurkat, MOLT4) and showed equivalent effects on genotoxic sensitivity and colony formation, as in AML. In addition, we found a two-fold increase in 7-AAD-assessed apoptosis, and a significantly reduced proliferation of CHD4 depleted cells in both T-ALL and AML cell lines. These effects are correlated with and possibly mediated by observed E2F1 depletion in CHD4 knockdown leukemia cell lines. Moreover, a five-fold reduction in the number of colonies of primary AML cells in methylcellulose-based media was observed. Similar to the findings in AML, MBD proteins showed redundancy in Jurkat T-ALL cells, whereby depletion of either MBD2 or MBD3 did not increase genotoxic sensitivity or reduce colony forming potential, but depletion of both together did so by a factor of up to three to five-fold respectively. Restoring MBD2 in U937 leukemia cells depleted of both MBD2 and MBD3 reversed increased genotoxic sensitization, demonstrating the on-target effects of MBD2 shRNA knockdown. We conclude that depletion of CHD4, or combined depletion of MBD2 and MBD3, produce equivalent beneficial phenotypes in both AML and T-ALL cell lines through disruption of the NuRD complex. Thus, targeting the common coiled coil structural motif that links both MBD2 and MBD3 to the GATAD2-CHD4 chromatin remodeling component of the NuRD complex may offer promise for improved therapy in acute leukemias of both myeloid and lymphoblastic origin. Citation Format: Javeria Aijaz, Shengzhe Shang, David C. Williams, Gordon D. Ginder. NuRD dpendent survival of acute leukemia cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 82.

The Nucleosome Remodeling and Deacetylase (NuRD) complex uniquely combines both deacetylase and remodeling enzymatic activities in a single macromolecular complex. The methyl-CpG–binding domain 2 and 3 (MBD2 and MBD3) proteins provide a critical structural link between the deacetylase and remodeling components, while MBD2 endows the complex with the ability to selectively recognize methylated DNA. Hence, NuRD combines three major arms of epigenetic gene regulation. Research over the past few decades has revealed much of the structural basis driving formation of this complex and started to uncover the functional roles of NuRD in epigenetic gene regulation. However, we have yet to fully understand the molecular and biophysical basis for methylation-dependent chromatin remodeling and transcription regulation by NuRD. In this review, we discuss the structural information currently available for the complex, the role MBD2 and MBD3 play in forming and recruiting the complex to methylated DNA, and the biological functions of NuRD.

BACKGROUND:MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA-protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. RESULTS:We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. CONCLUSIONS:Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.

Evolution has converged on cation-π interactions for recognition of quaternary alkyl ammonium groups such as trimethyllysine (Kme3). While computational modelling indicates that Trp provides the strongest cation-π interaction of the native aromatic amino acids, there is limited corroborative data from measurements within proteins. Herein we investigate a Tyr to Trp mutation in the binding pocket of the HP1 chromodomain, a reader protein that recognizes Kme3. Binding studies demonstrate that the Trp-mediated cation-π interaction is about -5 kcal mol-1 stronger, and the Y24W crystal structure shows that the mutation is not perturbing. Quantum mechanical calculations indicate that greater enthalpic binding is predominantly due to increased cation-π interactions. NMR studies indicate that differences in the unbound state of the Y24W mutation lead to enthalpy-entropy compensation. These results provide direct experimental quantification of Trp versus Tyr in a cation-π interaction and afford insight into the conservation of aromatic cage residues in Kme3 reader domains.