Analysis of the complex between MBD2 and the histone deacetylase core of NuRD reveals key interactions critical for gene silencing.

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.

MBD2a-NuRD binds to the methylated γ-globin gene promoter and uniquely forms a complex required for silencing of HbF expression.

During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2-NuRD. In this report, we present direct evidence that MBD2-NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a-NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine-binding domain (MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a-NuRD, PRMT5, and DNA methylation in HbF silencing.

Targeting the PLK1-FOXO1 pathway as a novel therapeutic approach for treating advanced prostate cancer.

The forkhead box protein O1 (FOXO1) is considered to be a key tumor suppressor due to its involvement in a broad range of cancer-related functions, including cellular differentiation, apoptosis, cell cycle arrest, and DNA damage. Given that inactivation of FOXO1 has been reported in many types of human cancer, we sought to investigate whether restoration of the pro-apoptotic activity of FOXO1 may be used as a new promising strategy for cancer treatment. Our previous study revealed that Polo-like kinase 1 (PLK1), a serine/threonine kinase that is essential for cell cycle progression, is a novel and major regulator of FOXO1 in the late phases of the cell cycle. Here, we provided evidence that PLK1-dependent phosphorylation of FOXO1 induces its nuclear exclusion and negatively regulates FOXO1's transcriptional activity in prostate cancer (PCa). Blocking the PLK1-dependant phosphorylation of FOXO1 restored the pro-apoptotic function of FOXO1 in PCa. Combining PLK1 inhibition with nocodazole (to induce mitotic arrest) had synergistic antitumor effects in vitro, with minimal effect on normal prostate epithelial cells. These findings shed light on a novel approach to reactivate apoptotic pathways in advanced PCa and support targeting PLK1-FOXO1 pathways as a novel approach for treating advanced PCa.

Expression of recombinant human lysozyme in bacterial artificial chromosome transgenic mice promotes the growth of Bifidobacterium and inhibits the growth of Salmonella in the intestine.

Targeted gene modification is a novel intervention strategy to increase disease resistance more quickly than traditional animal breeding. Human lysozyme, a natural, non-specific immune factor, participates in innate immunity, exerts a wide range of antimicrobial activities against pathogens, and has immuneregulatory effects. Therefore, it is a candidate gene for improved disease resistance in animals. In this study, we successfully generated a transgenic mouse model by microinjecting a modified bacterial artificial chromosome containing a recombinant human lysozyme (rhLZ) gene into the pronuclei of fertilized mouse embryos. rhLZ was expressed in serum, liver, spleen, lung, kidney, stomach, small intestine, and large intestine but not in milk. rhLZ protein concentrations in the serum of transgenic mice ranged from 2.09 to 2.60 mg/l. To examine the effect of rhLZ on intestinal microbiota, total aerobes, total anaerobes, Clostridium, Enterococcus, Streptococcus, Salmonella, Escherichia coli, Staphylococcus, Bifidobacterium, and Lactobacillus were measured in the intestines of transgenic and wild type mice. Results showed that Bifidobacteria were significantly increased (p < 0.001), whereas Salmonella were significantly decreased (p < 0.001) in transgenic mice compared to wild type mice. Our study suggests that rhLZ expression is a potential strategy to increase animal disease resistance.

Production of transgenic-cloned pigs expressing large quantities of recombinant human lysozyme in milk.

Human lysozyme is a natural non-specific immune factor in human milk that plays an important role in the defense of breastfed infants against pathogen infection. Although lysozyme is abundant in human milk, there is only trace quantities in pig milk. Here, we successfully generated transgenic cloned pigs with the expression vector pBAC-hLF-hLZ-Neo and their first generation hybrids (F1). The highest concentration of recombinant human lysozyme (rhLZ) with in vitro bioactivity was 2759.6 ± 265.0 mg/L in the milk of F0 sows. Compared with wild-type milk, rhLZ milk inhibited growth of Escherichia coli K88 during the exponential growth phase. Moreover, rhLZ in milk from transgenic sows was directly absorbed by the intestine of piglets with no observable anaphylactic reaction. Our strategy may provide a powerful tool for large-scale production of this important human protein in pigs to improve resistance to pathogen infection.

High-level recombinant human lysozyme expressed in milk of transgenic pigs can inhibit the growth of Escherichia coli in the duodenum and influence intestinal morphology of sucking pigs.

Lysozyme is often used as a feed additive and acts as an antimicrobial protein that enhances immune function and defends against pathogenic bacteria in pigs. In this study, we genetically added recombinant human lysozyme (rhLZ) to sow milk by somatic cell nuclear transfer and investigated whether the presence of recombinant human lysozyme can influence intestinal microbiota and morphology in sucking pigs. We generated transgenic cloned pigs and the first-generation hybrids (F1) produced high levels of rhLZ in milk. The average concentration of rhLZ was 116.34 ± 24.46 mg/L in the milk of F1 sows, which was 1500-fold higher than that of the native pig lysozyme. In vitro, it was demonstrated that rhLZ in milk of transgenic pigs had enzyme levels at 92,272 ± 26,413 U/mL. In a feeding experiment, a total of 40 newborn piglets were nursed by four transgenic sows and four sibling non-transgenic sows (F1), with five piglets per gilt. The piglets were allowed to nurse for 21 days and the sow milk was the only source of nutrition for the piglets. All piglets were slaughtered on postnatal day 22. Six types of bacteria were cultured and analyzed to detect the impact of rhLZ on gut microbiota. The number of Escherichia coli in the duodenum of piglets reared by transgenic sows was significantly decreased (p<0.001) and their villus height to crypt depth ratio in the intestine were increased due to the significant decrease of crypt depth in the duodenum, jejunum, and ileum (p<0.001). Together, we successfully generated rhLZ transgenic cloned pigs and elevated lysozyme level in nuring piglets. The results of the feeding experiments demonstrated that rhLZ-enhanced milk can inhibit the growth of E. coli in the duodenum and positively influence intestinal morphology without adversely affecting weight gain or piglet growth.

High-level expression of bioactive recombinant human lysozyme in the milk of transgenic mice using a modified human lactoferrin BAC.

Transgenesis has been used for expressing human lysozyme (hLZ) in the milk of livestock to improve their disease resistance. Here we describe a human lactoferrin (hLF) BAC as a candidate vector for high-level expression of hLZ in the milk of transgenic mice. Using recombineering, hLF genomic DNA in the hLF BAC was replaced by the hLZ gene (from the ATG start codon to the TAA stop codon), and flanking regions of the hLF gene (a 90-kb 5' and a 30-kb 3') were used as transcriptional control elements for hLZ expression. When this construct was used to generate transgenic mice, rhLZ was highly expressed in the milk of four transgenic mouse lines (1.20-1.76 g/L), was expressed at a lower level in one additional line (0.21 g/L). rhLZ from the milk of these transgenic mice exhibited the same antibacterial activity as native hLZ. Our results suggest a potential approach for producing large amounts of hLZ in the milk of livestock.