Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome.

The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.

Molecular Topology of RNA Polymerase I Upstream Activation Factor.

Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.

Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly.

RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre-initiation complex. Upstream Activating Factor (UAF) is one of two multi-subunit complexes that regulate pre-initiation complex formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. We found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3-H4 tetramer-like subcomplex.

Super elongation complex contains a TFIIF-related subcomplex.

Super elongation complex (SEC) belongs to a family of RNA polymerase II (Pol II) elongation factors that has similar properties as TFIIF, a general transcription factor that increases the transcription elongation rate by reducing pausing. Although SEC has TFIIF-like functional properties, it apparently lacks sequence and structural homology. Using HHpred, we find that SEC contains an evolutionarily related TFIIF-like subcomplex. We show that the SEC subunit ELL interacts with the Pol II Rbp2 subunit, as expected for a TFIIF-like factor. These findings suggest a new model for how SEC functions as a Pol II elongation factor and how it suppresses Pol II pausing.

Beta-cell-tropin is associated with short-term stimulation of food intake in chicks.

Beta-cell-tropin, a peptide derived from adrenocorticotropic hormone, is an insulin secretagogue. When centrally injected, it increases food intake in rats, but its appetite-associated effects have not been reported in any other species. Thus, the present study was designed to evaluate the effects of central beta-cell-tropin on appetite-associated parameters in an alternative vertebrate model, the chick. Central injection of 2 or 4 nmol beta-cell-tropin increased food intake for 60 min. Whole hypothalamus was collected at 60 min post-injection, and real-time PCR performed to measure mRNA abundance of agouti-related peptide, corticotropin releasing factor, galanin, melanin concentrating hormone, neuropeptide Y, orexin, prohormone convertase 2, pro-opiomelanocortin, peroxisome proliferator-activated receptor γ, urotensin 2, and visfatin, not one of which were affected by beta-cell-tropin treatment. Results demonstrate that beta-cell-tropin is associated with short-term stimulation of food intake.

The threshold of insulin-induced hypophagia is lower in chicks selected for low rather than high juvenile body weight.

Chicks genetically selected for low juvenile body weight had a lower threshold of central insulin-induced decreased food and water intake and whole blood glucose concentration than those selected for juvenile high body weight. Plasma corticosterone concentration was increased but not differently between lines. Therefore, selection may have affected insulin sensitivity which may have then contributed to their hypo- and hyperphagia and differential body weights.

Both calcitonin and calcitonin gene-related peptides' thresholds of hypophagia are considerably lower in chicks selected for high rather than low juvenile body weight.

Effects of intracerebroventricular (ICV) injection of calcitonin (CT) and calcitonin gene-related peptide (CGRP) on food and water intake were measured in two lines of White Plymouth Rock chickens from a common base population that have undergone long-term divergent selection for either low (LWS) or high (HWS) juvenile body weight. These lines contain anorexic and obese individuals and serve as models for hypo- and hyperphagia. For both ICV injection of CT and CGRP, line HWS responded to a lower dose with decreased food intake than did line LWS. Both peptides were also associated with reduced water intake in both lines. Although plasma glucose concentrations were inherently different between lines, neither CT nor CGRP affected these levels. Comprehensive behavior analyses were conducted and only the number of food pecks was differentially suppressed between lines after both CT and CGRP injection. Thus, the selection program may have caused alterations in the endogenous CT and CGRP systems that synergistically, with other neurotransmitter systems, contribute to the role of food intake on the differential body weights between these lines.

The threshold of amylin-induced anorexia is lower in chicks selected for low compared to high juvenile body weight.

Chicks that have undergone long-term selection for low body weight responded to intracerebroventricular amylin injection with reduced food intake at a dose considerably lower and with a greater magnitude suppression than those selected for high body weight. Behaviors unrelated to ingestion were not affected. These data support the thesis of correlated amylin system responses to selection for low or high body weight, with possible implications to other species.

Gamma(2)-melanocyte stimulating hormone decreases food intake in chicks.

The role of gamma melanocyte stimulating hormone (gamma-MSH) in appetite regulation is controversial in mammals and to our knowledge unreported within the avian class. Thus, the present study was designed to determine the effects of intracerebroventricularly (i.c.v.) administered gamma2-MSH on food intake using Cobb-500 chicks as models. In Experiment 1, chicks that received i.c.v. gamma2-MSH decreased their food intake throughout the 180 min observation period and plasma glucose concentration was not affected. Water intake was also decreased in i.c.v. gamma2-MSH-treated chicks, but only from 30 to 90 min post-injection. In Experiment 2, food pecking efficiency was decreased in i.c.v. gamma2-MSH-treated chicks and the amount of time spent sitting was increased. Other behaviors were not significantly affected by i.c.v. gamma2-MSH including distance traveled, the number of jumps, escape attempts, defecations, food pecks, exploratory pecks, and the amount of time spent standing, preening, perching, or in deep rest. These data suggest that gamma2-MSH is associated with anorexigenic effects and because of gamma-MSH's selectivity, implicates the melanocortin 3 receptor in appetite regulation.

Differential feed intake responses to central corticotrophin releasing factor in lines of chickens divergently selected for low or high body weight.

Effects of intracerebroventricular (ICV) injection of corticotrophin releasing factor (CRF) on feed intake were evaluated in two lines of White Plymouth Rock chickens that have been selected from a common base population for high (HWS) or low (LWS) juvenile body weight. Both lines responded with reduced feed intake after ICV CRF; however, the threshold of response was lower in line LWS than HWS. Additionally, the effects of two receptor antagonists, astressin and alpha-helical CRF (9-41; alpha-CRF), and the effect of CRF fragment 6-33, (which displaces CRF from its binding protein), were evaluated in these lines. Although all three antagonists increased feed intake in line LWS but not line HWS, they attenuated the appetite-reducing effects of CRF only in line HWS. Peripheral plasma corticosterone concentrations after an acute stressor were higher in line LWS than in line HWS. These data support the thesis of correlated responses in the CRF system to selection for high or low juvenile body weight. These differences may contribute to differential feed intake, and hence altered body weights.

Amylin causes anorexigenic effects via the hypothalamus and brain stem in chicks.

This study was conducted to determine the effects of amylin on appetite-related processes in chicks. Broiler chicks were centrally and peripherally injected with amylin, and feed and water intake were quantified. Feed intake was reduced after both central and peripheral amylin, but water intake was not affected. To determine if the hypothalamus and brainstem were involved in the anorexigenic effect, chicks were centrally and peripherally injected with amylin, and c-Fos immunoreactivity was quantified in the lateral hypothalamus (LH), ventromedial hypothalamus (VMH), area postrema (AP) and the nucleus of the solitary tract (NTS). Amylin decreased c-Fos immunoreactivity in the LH, did not affect the VMH, and increased c-Fos immunoreactivity in the AP and NTS. To determine if alimentary transit time was affected, chicks received central amylin and were gavaged with chicken feed slurry containing a visible marker. Amylin-treated chicks had increased alimentary canal transit time. Chicks also responded to central amylin with increased anxiety-related behaviors and increased plasma corticosterone concentration. These results demonstrate that amylin affects feeding, alimentary canal transit, and behavior through hypothalamic and brainstem mechanisms in chicks.

Central alpha-melanocyte stimulating hormone attenuates behavioral effects of neuropeptide Y in chicks.

This experiment was conducted to determine the effects of central alpha-melanocyte stimulating hormone (alpha-MSH) and its interaction with neuropeptide Y (NPY) on ingestive and non-ingestive behaviors in chicks. Chicks received intracerebroventricular injections of either 0, 0.12 nM alpha-MSH, 0.06 nM NPY, or 0.12 nM alpha-MSH+0.06 nM NPY. Immediately following injection, chicks were placed in an observation arena and the number of steps, jumps, feed pecks, drinks, exploratory pecks, escape attempts, the total distance traveled, and the amount of time spent standing, sitting, sleeping, and preening were monitored for 60 min. Chicks treated with NPY consumed 69% more feed than controls whereas alpha-MSH-treated chicks consumed 71% less. Feed intake of the NPY+alpha-MSH groups was similar to alpha-MSH-treated chicks at 66% less than aCSF-treated chicks. Differences in pecking were found and followed a similar pattern as feed intake. All treatments increased the amount of time chicks were in a sitting posture, and the alpha-MSH+NPY group spent more time sitting than alpha-MSH and NPY alone. The sitting response after alpha-MSH+NPY treatment was similar to the alpha-MSH group but not the NPY group. Other behaviors were not affected by treatment. Thus, we conclude that alpha-MSH, at a concentration that causes a similar magnitude decrease in feed intake as NPY increases feed intake, is a more potent appetite-related signal than NPY. alpha-MSH causes behavioral effects that may secondarily affect feed intake at a low magnitude and may modulate the behavioral effects of NPY in chicks, contributing to the overall effect on feed intake.