SUMMARY Upon androgen stimulation, PKN1-mediated histone H3 threonine 11 phosphorylation (H3T11P) promotes AR target genes activation. However, the underlying mechanism is not completely understood. Here, we show that WDR5, a subunit of the SET1/MLL complex, interacts with H3T11P and this interaction facilitates the recruitment of the MLL1 complex and subsequent H3K4 trimethylation (H3K4me3). Using ChIP-seq, we find that androgen stimulation results in a six-fold increase in the number of H3T11P-marked regions and induces WDR5 colocalization to one third of H3T11P-enriched promoters, thus establishing a genome-wide relationship between H3T11P and recruitment of WDR5. Accordingly, PKN1 knock-down or chemical inhibition severely blocks WDR5 association and H3K4me3 on AR target genes. Finally, WDR5 is critical in prostate cancer cell proliferation, and is hyperexpressed in human prostate cancers. Together, these results identify WDR5 as a critical epigenomic integrator of histone phosphorylation and methylation and a major driver of androgen-dependent prostate cancer cell proliferation.

Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.transcription regulation | cellular signaling | protein-protein interaction

The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions in a single active site. PchB physiologically produces salicylate and pyruvate from isochorismate for ultimate incorporation of the salicylate into the siderophore pyochelin. PchB also produces prephenate from chorismate, most likely due to structural homology to the Escherchia coli chorismate mutase. The molecular basis of catalysis among enzymatic pericyclic reactions is a matter of debate, one view holding that catalysis may be derived from electrostatic transition state stabilization and the opposing view that catalysis is derived from the generation of a reactive substrate conformation. Mutant forms of PchB were generated by site-directed mutagenesis at the site (K42) hypothesized to be key for electrostatic transition state stabilization (K42A, K42Q, K42E, and K42H). The loop containing K42 is mobile, and a mutant to slow loop dynamics was also designed (A43P). Finally, a previously characterized mutation (I87T) was also produced. Circular dichroism was used to assess the overall effect on secondary structure as a result of the mutations, and X-ray crystallographic structures are reported for K42A with salicylate and pyruvate bound and for apo-I87T. The data illustrate that the active site architecture is maintained in K42A-PchB, which indicates that differences in activity are not caused by secondary structural changes or by differences in active site loop conformation but rather by the chemical nature of this key residue. In contrast, the I87T structure demonstrates considerable mobility, suggesting that loop dynamics and conformational plasticity may be important for efficient catalysis. Finally, the mutational effects on k(cat) provide evidence that the two activities of PchB are not covariant and that a single hypothesis may not provide a sufficient explanation for catalysis.

The differences in physiological and immunological parameters and pathological damage to organ tissues exposed to chronic heat stress provide the basis for evaluating heat resistance of different chicken breeds (white recessive rock [WRR] and The Lingshan [LS]). Ninety broilers of each breed were divided equally into a chronic heat stress group and a no heat stress group. The effects of chronic heat stress on the physiological and immunological parameters of broilers were analyzed using flow cytometry, ELISA, RT-qPCR, etc. Under heat stress conditions: (1) H and H/L values were significantly increased (P < 0.01) in the 2 breeds, and were higher in the WRR broilers than in the LS broilers at a late stage (P < 0.05). Although the corticosterone levels were also significantly increased (P < 0.01) in both breeds, they were lower in the 49 d WRR broilers than in the LS broilers (P < 0.01). The number of leukocytes were significantly increased in the 49 d WRR broilers (P < 0.01), whereas the number of CD3+, CD8+ cells, and erythrocytes were significantly reduced (P < 0.05). A significantly (P < 0.01) lower number of CD3+, CD4+ T-lymphocytes, and CD4+/CD8+ were present in WRR compared to that in the LS broilers. (2) The HSP70 transcript was significantly increased in the WRR broilers (P < 0.01), and was higher than the level in the LS broilers. The expression level of HSP70 protein was significantly (P < 0.05) increased in WRR broilers. (3) The WRR broilers developed cardiac and leg muscle inflammatory cellular hyperplasia and local inflammatory lesions, as well as cerebral meningitis and inflammatory hyperplasia of the brain tissue. The LS broilers developed mild cerebral inflammatory hyperplasia and mild inflammatory cellular proliferation in the leg muscle. In conclusion, under heat stress conditions, the relative physiological and immunological parameters were worse in the WRR broilers than in the LS broilers. The WRR broilers showed poor heat tolerance as evidenced from the expression of HSP70 and the extent of histopathological damages.

No abstract

The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions, demonstrating the eponymous activity and also chorismate mutase activity. The thermodynamic parameters for these enzyme-catalyzed activities, as well as the uncatalyzed isochorismate decomposition, are reported from temperature dependence of kcat and kuncat data. The entropic effects do not contribute to enzyme catalysis as expected from previously reported chorismate mutase data. Indeed, an entropic penalty for the enzyme-catalyzed mutase reaction (ΔS‡ = -12.1 ± 0.6 cal/molK) is comparable to that of the previously reported uncatalyzed reaction, whereas that of the enzyme-catalyzed lyase reaction (ΔS‡ = -24.3 ± 0.6 cal/molK) is larger than that of the uncatalyzed lyase reaction (-15.77 ± 0.02 cal/molK) documented here. With the assumption that chemistry is rate-limiting, we propose that a reactive substrate conformation is formed upon loop closure of the active site and that ordering of the loop contributes to the entropic penalty for converting the enzyme substrate complex to the transition state.

Excessive abdominal fat deposition is an issue with general concern in broiler production, especially for Chinese native chicken breeds. A high-fat diet (HFD) can induce body weight gained and excessive fat deposition, and genes and pathways participate in fat metabolism and adipogenesis would be influenced by HFD. In order to reveal the main genes and pathways involved in chicken abdominal fat deposition, we used HFD and normal diet (ND) to feed a Chinese native chicken breed, respectively. Results showed that HFD can increase abdominal fat deposition and induce adipocyte hypertrophy. Additionally, we used RNA-sequencing to identify the differentially expressed genes (DEGs) between HFD and ND chickens in liver and abdominal fat. By analyzed these DEGs, we found that the many DEGs were enriched in fat metabolism related pathways, such as peroxisome proliferator-activated receptor (PPAR) signaling, fat digestion and absorption, extracellular matrix (ECM)-receptor interaction, and steroid hormone biosynthesis. Notably, the expression of insulin-like growth factor II mRNA binding protein 1 (IGF2BP1), which is a binding protein of IGF2 mRNA, was found to be induced in liver and abdominal fat by HFD. Ectopic expression of IGF2BP1 in chicken liver-related cell line Leghorn strain M chicken hepatoma (LMH) cell revealed that IGF2BP1 can regulate the expression of genes associated with fatty acid metabolism. In chicken preadipocytes (ICP cell line), we found that IGF2BP1 can promote adipocyte proliferation and differentiation, and the lipid droplet content would be increased by overexpression of IGF2BP1. Taken together, this study provides new insights into understanding the genes and pathways involved in abdominal fat deposition of Chinese native broiler, and IGF2BP1 is an important candidate gene for the study of fat metabolism and adipogenesis in chicken.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that has a similar replication cycle to multiple viruses and therefore can be used as a model system for viral entry into host cells. However, there are few reports on the genes or microRNAs (miRNAs) that are responsible for the replication of ALV-J. Our previous miRNA and RNA sequencing data showed that the expression of miR-34b-5p was significantly upregulated in ALV-J-infected chicken spleens compared to non-infected chicken spleens, but melanoma differentiation-associated gene 5 (MDA5) had the opposite expression pattern. In this study, a dual-luciferase reporter assay showed that MDA5 is a direct target of miR-34b-5p. In vitro, overexpression of miR-34b-5p accelerated the proliferation of ALV-J-infected cells by inducing the progression from G2 to S phase and it promoted cell migration. Ectopic expression of MDA5 inhibited ALV-J-infected cell proliferation, the cell cycle and cell migration, and knockdown of MDA5 promoted proliferation, the cell cycle and migration. In addition, during ALV-J infections, MDA5 can detect virus invasion and it triggers the MDA5 signaling pathway. MDA5 overexpression can activate the MDA5 signaling pathway, and thus it can inhibit the mRNA and protein expression of the ALV-J env gene and it can suppress virion secretion. In contrast, in response to the knockdown of MDA5 by small interfering RNA (siRNA) or an miR-34b-5p mimic, genes in the MDA5 signaling pathway were significantly downregulated (P < 0.05), but the mRNA and protein expression of ALV-J env and the sample-to-positive ratio of virion in the supernatants were increased. This indicates that miR-34b-5p is able to trigger the MDA5 signaling pathway and affect ALV-J infections. Together, these results suggest that miR-34b-5p targets MDA5 to accelerate the proliferation and migration of ALV-J-infected cells, and it promotes ALV-J replication, via the MDA5 signaling pathway.