Biology subjects

Williams, D. C.

Publications and source records attributed to Williams, D. C..

8 recordsLinked to original sources

bioRxiv2026

Structure-guided targeting of the GATAD2A-CHD4 interaction within the MBD2-NuRD complex results in high levels of HbF in adult erythroid cells

Fetal hemoglobin (HbF) expression is silenced postnatally in adult erythroid cells. Sufficiently increased expression of HbF has been shown to overcome the pathophysiologic sequelae of both sickle cell disease and beta-thalassemia. As the MBD2a-NuRD chromatin remodeling complex is required for silencing of HbF, the present studies were aimed at exploring a potential therapeutic approach for disrupting this complex. AlphaFold 3 and a recent crystal structure were employed to predict the critical interaction domains linking GATAD2A in the histone deacetylase core subcomplex (HDCC) of NuRD and the CHD4 ATPase which has been shown to be required for silencing of the fetal gamma-globin (HBG) genes. The two predicted critical domains, the CR2 helical domain of GATAD2A and the C-terminal domains 1 and 2 (C1b and C2ab) of CHD4, were validated by in vitro biophysical studies. Mutation of two amino acids in the CR2 helical domain of the endogenous GATAD2A gene in HUDEP-2 cells resulted in dissociation of CHD4, loss of repressive chromatin over the HBG promoter and ~40% HbF levels compared to < 1% in control cells. Strikingly, enforced expression of a peptide containing the helical portion of the CR2 domain of GATAD2A in both HUDEP-2 cells and primary adult erythroid cells resulted in high levels of HbF, with up to ~75% HbF compared to mutant peptide control level of ~9% in the latter without perturbing erythroid differentiation. These results suggest that targeting the critical interaction domains of GATAD2A and CHD4 with a macrocyclic peptide or small molecule may lead to much needed small molecule therapeutics for sickle cell disease. Key PointsAssociation of CHD4 with the HDCC core of the MBD2-NuRD chromatin remodeling complex is required for silencing of HbF expression in adult human erythroid cells Genetic alteration or enforced peptide expression of a critical helical domain of GATAD2A results in dissociation of CHD4 from the MBD2-NuRD complex and high-level expression of HbF.

molecular biology↗

bioRxiv2026

Macrocycle screening against the C-terminal region of CHD4 uncovers its role as an interaction hub in the formation of the nucleosome remodeling and deacetylase complex

The Nucleosome Remodeling and Deacetylase complex (NuRD) plays a key role in regulating hemoglobin expression in adult erythroid cells. Selectively disrupting this complex potently induces the expression of fetal hemoglobin, a proven therapeutic strategy for treating beta-hemoglobinopathies such as sickle cell anemia. In these studies, we have used mRNA display to identify small macrocyclic peptides that inhibit the interaction between two core components of NuRD, the SANT-SLIDE domain of CHD4 and the CR2 domain of GATAD2A. In addition, the screen suggested a second binding site on the CHD4 domain. Based on this observation, we hypothesized and confirmed that CDK2AP1 bound to this region of CHD4, leading us to purify and determine the structure of the ternary complex between CHD4, GATAD2A, and CDK2AP1. The results of our studies show that the SANT-SLIDE domain of CHD4 functions as a critical interaction hub in the formation of NuRD and suggest a strategy to block NuRD function for therapy.

biophysics↗

bioRxiv2024

Ethograms reveal a fear conditioned visual cue to organize diverse behaviors in rats

Recognizing and responding to threat cues is essential to survival. In rats, freezing is the most common behavior measured. Previously we demonstrated a threat cue can organize diverse behaviors (Chu et al., 2024). However, the experimental design of Chu et al. (2024) was complex and the findings descriptive. Here, we gave female and male Long Evans rats simple paired or unpaired presentations of a light and foot shock (8 total) in a conditioned suppression setting, using a range of shock intensities (0.15, 0.25, 0.35 or 0.5 mA). We found that conditioned suppression was only observed at higher foot shock intensities (0.35 mA and 0.5 mA). We constructed comprehensive, temporal ethograms by scoring 22,272 frames of behavior for 12 mutually exclusive behavior categories in 200 ms intervals around cue presentation. A 0.5 mA and 0.35 mA shock-paired visual cue suppressed reward seeking, rearing and scaling, as well as light-directed rearing and light-directed scaling. The shock-paired visual paired cue further elicited locomotion and freezing. Linear discriminant analyses showed that ethogram data could accurately classify rats into paired and unpaired groups. Considering the complete ethogram data produced superior classification than considering subsets of behaviors. The results demonstrate diverse threat-elicited behaviors - in a simple Pavlovian fear conditioning design - containing sufficient information to distinguish the fear learning status of individual rats.

neuroscience↗

bioRxiv2023

Genome Sequence and Annotation the B3 Mycobacteriophage Phayeta

Phayeta is a siphovirus extracted from soil near Myrtle Beach, South Carolina which infects Mycobacterium smegmatis. Annotation of the 68,700 base-pair circularly permuted genome identified 104 predicted protein-encoding genes, 36 of which have functional assignments.

genomics↗

bioRxiv2022

A fear conditioned cue orchestrates a suite of behaviors

Pavlovian fear conditioning has been extensively used to study the behavioral and neural basis of defensive systems. In a typical procedure, a cue is paired with foot shock, and subsequent cue presentation elicits freezing, a behavior theoretically linked to predator detection. Studies have since shown a fear conditioned cue can elicit locomotion, a behavior that - in addition to jumping, and rearing - is theoretically linked to imminent or occurring predation. A criticism of studies observing fear conditioned cue-elicited locomotion is that responding is non-associative. We gave 24 rats (12 female) Pavlovian fear discrimination over a baseline of reward seeking. The within-subjects procedure had full controls for associative learning, consisting of three cues predicting unique foot shock probabilities: danger (p=1), uncertainty (p=0.25), and safety (p=0). TTL-triggered cameras captured 5 behavior frames/s prior to and during cue presentation. We scored 86,400 frames for nine discrete behaviors spanning reward, passive fear, and active fear. Temporal ethograms show that a fear conditioned cue elicits locomotion, jumping, and rearing that is maximal towards cue offset, when foot shock is imminent. A fear conditioned cue further suppresses reward-related behavior, and elicits freezing in a sex-specific manner. The differing temporal profiles and independent expression of these behaviors reveal a fear conditioned cue to orchestrate a rich and intricate suite of behaviors.

animal behavior and cognition↗

bioRxiv2022

MEIG1/PACRG associated and non-associated functions of axonemal dynein light intermediate polypeptide 1 (DNALI1) in mammalian spermatogenesis

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 statementAxonemal 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.

cell biology↗

bioRxiv2020

A Bacterial Signaling Network Controls Antibiotic Resistance by Regulating Anaplerosis of 2-oxoglutarate

Antibiotic resistance has become a global threat. In addition to acquiring resistance via horizontal gene transfer, bacteria can evade killing by temporarily modifying their cell envelope to prevent antibiotic-bacterial interactions. A critical gap in knowledge is how bacteria balance the metabolic needs of altering the cell envelope with the constant need to generate energy. Cross-regulation between two signaling networks in Escherichia coli increases resistance to positively charged antibiotics. We show that increased resistance is supported by metabolic re-wiring controlled by the QseB transcription factor. QseB controls the increase in 2-oxoglutarate required for lipid A modification, by upregulating three anaplerotic pathways that feed acetyl Co-A, succinate and fumarate into the TCA cycle. Exogenous addition of 2-oxoglutarate restores antibiotic resistance in the qseB deletion mutant. Antibiotic resistant clinical isolates bear mutations within QseB-mediated anaplerotic pathways. These findings are significant, because they uncover a previously unknown mechanism of metabolic control of antibiotic resistance.

microbiology↗

bioRxiv2020

The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture

The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for development in complex animals but has been refractory to biochemical analysis. We present the first integrated analysis of the architecture of the native mammalian NuRD complex, combining quantitative mass spectrometry, covalent cross-linking, protein biochemistry and electron microscopy. NuRD is built around a 2:2:4 pseudo-symmetric deacetylase module comprising MTA, HDAC and RBBP subunits. This module interacts asymmetrically with a remodeling module comprising one copy each of MBD, GATAD2 and CHD subunits. The previously enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the ATP-dependent CHD remodeler. Unexpectedly, the MTA-MBD interaction acts as a point of functional switching. The transcriptional regulator PWWP2A modulates NuRD assembly by competing directly with MBD for binding to the MTA-HDAC-RBBP subcomplex, forming a moonlighting PWWP2A-MTA-HDAC-RBBP complex that likely directs deacetylase activity to PWWP2A target sites. Taken together, our data describe the overall architecture of the intact NuRD complex and reveal aspects of its structural dynamics and functional plasticity.

biochemistry↗