Mutations truncating the EP300 acetylase in human cancers.

The EP300 protein is a histone acetyltransferase that regulates transcription via chromatin remodelling and is important in the processes of cell proliferation and differentiation. EP300 acetylation of TP53 in response to DNA damage regulates its DNA-binding and transcription functions. A role for EP300 in cancer has been implied by the fact that it is targeted by viral oncoproteins, it is fused to MLL in Leukaemia and two missense sequence alterations in EP300 were identified in epithelial malignancies. Nevertheless, direct demonstration of the role of EP300 in tumorigenesis by inactivating mutations in human cancers has been lacking. Here we describe EP300 mutations, which predict a truncated protein, in 6(3%) of 193 epithelial cancers analysed. Of these six mutations, two were in primary tumours (a colorectal cancer and a breast cancer) and four were in cancer cell lines (colorectal, breast and pancreatic). In addition, we identified a somatic in-frame insertion in a primary breast cancer and missense alterations in a primary colorectal cancer and two cell lines (breast and pancreatic). Inactivation of the second allele was demonstrated in five of six cases with truncating mutations and in two other cases. Our data show that EP300 is mutated in epithelial cancers and provide the first evidence that it behaves as a classical tumour-suppressor gene.

Sex differences in musculoskeletal injury epidemiology and subsequent loss of tactical readiness during Marine Corps Officer Candidates School.

INTRODUCTION: The US Marine Corps (USMC) Officer Candidates School (OCS) is a 10-week training course for Marine Officer Candidates (MOCs). OCS training is rigorous and demanding, which results in a high risk of musculoskeletal injuries (MSIs). The objective of this analysis was to describe MSIs among women and men during the USMC OCS at Quantico, Virginia, from September 2020 to November 2021. METHODS: This prospective cohort study assessed MSIs that occurred among 736 MOCs (women: 17.8% of sample, men: 82.2%). Data for the study were derived from routinely collected injury data by athletic trainers and physical therapists embedded within the training units. Injury incidence, event at the time of injury occurrence, anatomic location, injury type and disposition following injury were described. Fisher's exact tests were used to compare proportions of injured women and men. RESULTS: The cumulative injury incidence was higher among women (39.7%) compared with men (23.1%, p<0.001). When specific events associated with injuries were reported, most frequent events were the obstacle course (women: 20.9% of injuries, men: 12.9%) and the conditioning hike (women: 11.6%, men: 6.9%). Most injures affected the lower body (women: 67.4%, men: 70.8%). The most frequent body part injured was the lower leg (18.6%) in women and the knee (23.3%) in men. The most frequent injury type was strain (women: 39.5%, men: 24.3%), followed by sprain (women: 16.3%, men: 14.9%). A greater percentage of female (92.3%) compared with male MOCs (69.3%; p<0.001) were assigned light duty status following MSIs. CONCLUSIONS: Mitigation of injuries during OCS events such as the obstacle course and the conditioning hike needs further investigation. The high risk of overuse lower leg injuries among women and the higher incidence of injuries among women compared with men underscore the need for further investigation of modifiable sex-specific injury risk factors.

p300 is required for orderly G1/S transition in human cancer cells.

The role of the transcriptional coactivator p300 in cell cycle control has not been analysed in detail due to the lack of appropriate experimental systems. We have now examined cell cycle progression of p300-deficient cancer cell lines, where p300 was disrupted either by gene targeting (p300(-) cells) or knocked down using RNAi. Despite significant proliferation defects under normal growth conditions, p300-deficient cells progressed rapidly through G1 with premature S-phase entry. Accelerated G1/S transition was associated with early retinoblastoma (RB) hyperphosphorylation and activation of E2F targets. The p300-acetylase activity was dispensable since expression of a HAT-deficient p300 mutant reversed these changes. Co-immunoprecipitation showed p300/RB interaction occurs in vivo during G1, and this interaction has two peaks: in early G1 with unphosphorylated RB and in late G1 with phosphorylated RB. In vitro kinase assays showed that p300 directly inhibits cdk6-mediated RB phosphorylation, suggesting p300 acts in early G1 to prevent RB hyperphosphorylation and delay premature S-phase entry. Paradoxically, continued cycling of p300(-) cells despite prolonged serum depletion was observed, and this occurred in association with persistent RB hyperphosphorylation. Altogether, these results suggest that p300 has an important role in G1/S control, possibly by modulating RB phosphorylation.

Acetylation of importin-alpha nuclear import factors by CBP/p300.

Histone acetylases were originally identified because of their ability to acetylate histone substrates [1] [2] [3]. Acetylases can also target other proteins such as transcription factors [4] [5] [6] [7]. We asked whether the acetylase CREB-binding protein (CBP) could acetylate proteins not directly involved in transcription. A large panel of proteins, involved in a variety of cellular processes, were tested as substrates for recombinant CBP. This screen identified two proteins involved in nuclear import, Rch1 (human importin-alpha) and importin-alpha7, as targets for CBP. The acetylation site within Rch1 was mapped to a single residue, Lys22. By comparing the context of Lys22 with the sequences of other known substrates of CBP and the closely related acetylase p300, we identified G/SK (in the single-letter amino acid code) as a consensus acetylation motif. Mutagenesis of the glycine, as well as the lysine, severely impaired Rch1 acetylation, supporting the view that GK is part of a recognition motif for acetylation by CBP/p300. Using an antibody raised against an acetylated Rch1 peptide, we show that Rch1 was acetylated at Lys22 in vivo and that CBP or p300 could mediate this reaction. Lys22 lies within the binding site for a second nuclear import factor, importin-beta. Acetylation of Lys22 promoted interaction with importin-beta in vitro. Collectively, these results demonstrate that acetylation is not unique to proteins involved in transcription. Acetylation may regulate a variety of biological processes, including nuclear import.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein.

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

In vitro DNA binding activity of Fos/Jun and BZLF1 but not C/EBP is affected by redox changes.

The leucine zipper family of proteins have a DNA binding domain composed of a leucine zipper dimerisation interface and a basic DNA binding structure. We show here that redox changes affect the in vitro DNA binding ability of a select subset of leucine zipper proteins. The bacterially expressed DNA binding domains of Fos/Jun and BZLF1 are unable to bind DNA under non-reducing conditions whereas binding of the C/EBP DNA binding domain is unaffected. Sensitivity to redox state is due to the presence of a conserved cysteine residue in the basic DNA binding motif of Fos, Jun and BZLF1 but not C/EBP. Under non-reducing conditions an intermolecular disulphide bridge is formed between the cysteine residues of each basic motif within a dimer, which prevents DNA binding. We show that oxidation of these C residues can be achieved enzymatically, using glutathione peroxidase, and that DNA binding protects them from oxidation. These data raise the possibility that intracellular changes in the redox state may differentially regulate the activity of leucine zipper family members. In addition the loss of DNA binding activity under non-reducing conditions has implications for the purification methods used to isolate proteins of the leucine zipper family for structural analysis.

The putative tumour suppressor Fus-2 is an N-acetyltransferase.

Acetyltransferases are essential enzymes for a wide variety of cellular processes and mutations in acetyltransferase genes have been associated with the development of certain cancers. For this reason, we conducted a computerized sequence homology search for novel acetyltransferases. Here, we show that the putative tumour suppressor protein Fus-2 has homology to the catalytic domain of acetyltransferases. We demonstrate that Fus-2 can acetylate the N-terminus of proteins using a ping-pong mechanism and that it has a specificity for substrates. Consistent with other N-acetyltransferases, Fus-2 localizes to the cytoplasm, as shown by GFP-tag experiments. Since the Fus-2 gene maps to the chromosomal region 3p21.3, which contains at least one tumour suppressor gene, the N-acetyltransferase functions of Fus-2 may be relevant to its potential role in cancer.

The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A.

A yeast two-hybrid screen has identified HBP1 as a transcription factor capable of interacting with the pocket protein family. We show that HBP1 can interact with one of these, RB, both in vitro and in mammalian cells. Two distinct RB binding sites are present within HBP1--a high affinity binding site, mediated by an LXCXE motif and a separate low affinity binding site present within an activation domain. GAL4-fusion experiments indicate that HBP1 contains a masked activation domain. Deletion of two independent N- and C-terminal inhibitor domains unmasks an activation domain which is 100-fold more active than the full length protein. The released activation capacity is repressed by RB, p130 and p107. In addition, E1A can repress the activity of HBP1 via conserved region 1 sequences in a manner independent of the CBP co-activator. We show by stable expression in NIH3T3 cells that HBP1 has the capacity to induce morphological transformation of cells in culture.

Stimulation of c-Jun activity by CBP: c-Jun residues Ser63/73 are required for CBP induced stimulation in vivo and CBP binding in vitro.

The CBP protein mediates PKA induced transcription by binding to the PKA phosphorylated activation domain of CREB. Here we show that CBP also stimulates the activity of both c-Jun and v-Jun in vivo. The CREB binding domain of CBP is sufficient to contact to c-Jun in vitro. When this domain of CBP is linked to the activation domain of VP16 and expressed in vivo it stimulates c-Jun dependent transcription. Deletion analysis of c-Jun indicate that the CBP binding site is within the N-terminal activation domain. Loss of binding to CBP in vitro correlates with severely reduced transactivation capacity in vivo. Mutation of Ser63/73 in c-Jun, or the corresponding position in v-Jun (Ser36/46) leads to reduced binding to CBP in vitro and abolishes augmentation of transcription in vivo. These data are consistent with a mechanism by which CBP acts as a co-activator protein for Jun dependent transcription by interacting with the Jun N-terminal activation domain.

Inter- and intra-laminar connections of pyramidal cells in the neocortex.

The flow of excitation through cortical columns has long since been predicted by studying the axonal projection patterns of excitatory neurones situated within different laminae. In grossly simplified terms and assuming random connectivity, such studies predict that input from the thalamus terminates primarily in layer 4, is relayed 'forward' to layer 3, then to layers 5 and 6 from where the modified signal may exit the cortex. Projection patterns also indicate 'back' projections from layer 5 to 3 and layer 6 to 4. More recently it has become clear that the interconnections between these layers are not random; forward projections primarily contact specific pyramidal subclasses and intracortical back projections innervate interneurones. This indicates that presynaptic axons or postsynaptic dendrites are capable of selecting their synaptic partners and that this selectivity is layer dependent. For the past decade, we and others have studied pyramidal cell targeting in circuits both within, and between laminae using paired intracellular recordings with biocytin filling and have begun to identify further levels of selectivity through the preferential targeting of electrophysiologically and/or morphologically distinct pyramidal subtypes. Presented here, therefore, is a brief overview of current thinking on the layer and subclass specific connectivity of neocortical principle excitatory cells.

Postsynaptic pyramidal target selection by descending layer III pyramidal axons: dual intracellular recordings and biocytin filling in slices of rat neocortex.

Paired intracellular recordings in slices of adult rat neocortex with biocytin filling of synaptically connected neurons were used to investigate the pyramidal targets, in layer V, of layer III pyramidal axons. The time-course and sensitivity of excitatory postsynaptic potentials to current injected at the soma, and locations of close appositions between presynaptic axons and postsynaptic dendrites, indicated that the majority of contributory synapses were located in layer V. Within a "column" of tissue, radius < or = 250 microm, the probability that a randomly selected layer III pyramid innervated a layer V pyramid was 1 in 4 if the target cell was a burst firing pyramid with an apical dendritic tuft in layers II/I. If, however, the potential target was a regular spiking pyramid, the probability of connectivity was only 1 in 40, and none of the 13 anatomically identified postsynaptic layer V targets had a slender apical dendrite terminating in layers IV/III. Morphological reconstructions indicated that layer III pyramids select target layer V cells whose apical dendrites pass within 50-100 microm of the soma of the presynaptic pyramid in layer III and which have overlapping apical dendritic tufts in the superficial layers. The probability that a layer V cell would innervate a layer III pyramid lying within 250 microm of its apical dendrite was much lower (one in 58). Both presynaptic layer III pyramids and their large postsynaptic layer V targets could therefore access similar inputs in layers I/II, while small layer V pyramids could not. One prediction from the present data would be that neither descending layer V inputs to the striatum or thalamus, nor transcallosal connections would be readily activated by longer distance cortico-cortical "feedback" connections that terminated in layers I/II. These could, however, activate corticofugal pathways to the superior colliculus or pons, both directly and via layer III.

Double immunofluorescence, peroxidase labelling and ultrastructural analysis of interneurones following prolonged electrophysiological recordings in vitro.

Inhibitory hippocampal and neocortical interneurones comprise a physiologically, morphologically and neurochemically heterogenous cell population. To identify the roles each class of interneurone plays within a given circuit it is necessary to correlate the electrophysiological properties of individual cells with their neurochemistry and morphology at both the light and electron microscopic level. However, the optimal conditions required for any one part of the protocol typically compromise the results from another. We have developed a protocol which allows the neurochemical content, gross morphology and ultrastructure details of biocytin-filled neurones to be recovered following long, dual intracellular recordings in thick mature slices maintained in an interface recording chamber, helping define sub-populations which could not otherwise be determined. Dual immunofluorescence is performed by incubating the tissue in monoclonal and polyclonal antibodies simultaneously, prior to visualization of biocytin-labelling with precipitation of a peroxidase reaction product. By using a biotinylated anti-avidin D antibody (Vector Laboratories), the intensity of this precipitation can be enhanced further where necessary. It is envisaged that this protocol can not only help determine the neurochemical content of cells recorded in similar in vivo studies, but that the ability to amplify peroxidase labelling in poorly filled cells is also of interest.

BET protein inhibition shows efficacy against JAK2V617F-driven neoplasms.

Small molecule inhibition of the BET family of proteins, which bind acetylated lysines within histones, has been shown to have a marked therapeutic benefit in pre-clinical models of mixed lineage leukemia (MLL) fusion protein-driven leukemias. Here, we report that I-BET151, a highly specific BET family bromodomain inhibitor, leads to growth inhibition in a human erythroleukemic (HEL) cell line as well as in erythroid precursors isolated from polycythemia vera patients. One of the genes most highly downregulated by I-BET151 was LMO2, an important oncogenic regulator of hematopoietic stem cell development and erythropoiesis. We previously reported that LMO2 transcription is dependent upon Janus kinase 2 (JAK2) kinase activity in HEL cells. Here, we show that the transcriptional changes induced by a JAK2 inhibitor (TG101209) and I-BET151 in HEL cells are significantly over-lapping, suggesting a common pathway of action. We generated JAK2 inhibitor resistant HEL cells and showed that these retain sensitivity to I-BET151. These data highlight I-BET151 as a potential alternative treatment against myeloproliferative neoplasms driven by constitutively active JAK2 kinase.

Dynamic properties of excitatory synaptic connections involving layer 4 pyramidal cells in adult rat and cat neocortex.

To investigate the properties of excitatory connections between layer 4 pyramidal cells and whether these differed between rat and cat, paired intracellular recordings were made with biocytin filling in slices of adult neocortex. These connections were also compared with those from layer 4 spiny cells to layer 3 pyramids and connections between layer 3 pyramids. Connectivity ratios for layer 4 pyramid-pyramid pairs (1:14 cat, 1:18 rat) appeared lower than for the other types of connections studied in parallel, but excitatory postsynaptic potential (EPSP) amplitudes and time course were not significantly different either between species or across types of connection. Layer 4 pyramids targeted postsynaptic basal dendrites in both species, whether the pyramidal target was in layer 4 or layer 3. Within layer 4, relationships between mean EPSP amplitude, numbers of putative contacts, and distance between connected pairs indicated a rapid decline in connectivity strength with distance, equivalent to 3.4 mV and 10 synapses per 100 microm separation, from a maximum of 4 mV and 10 synapses at 0 microm. However, a subset, of burst-firing layer 4 pyramids, appeared to make no connections with other layer 4 spiny cells. Second EPSPs were depressed by 36% in rat and 28% in cat relative to first EPSPs at interspike intervals <15 ms. Subsequent EPSPs in brief trains were further depressed. Depression was predominantly presynaptic in origin. Recovery from depression could not be described adequately by a simple exponential for individual connections; it included peaks and troughs with periodicities of 10-15 ms. Complex relationships between the first 2 interspike intervals and third EPSP amplitude were also apparent in all connections so studied. Large third EPSPs followed specific combinations of first and second interspike intervals so that increasing, or decreasing, one without changing the other resulted in a smaller third EPSP. Finally, the outputs of layer 4 spiny cells to layer 3 exhibited partial recovery from depression during longer high-frequency trains, a property not apparent in the other connections studied.

Robust correlations between action potential duration and the properties of synaptic connections in layer 4 interneurones in neocortical slices from juvenile rats and adult rat and cat.

Many studies of cortical interneurones use immature rodent tissue, while many recordings in vivo are made in adult cats. To determine the extent to which interneuronal circuitry studied with one approach can transfer to another, we compared layer 4 interneurones and their local connections across two age groups and two species and with similar connections in layers 3 and 5, using two common recording techniques: dual whole cell recordings at 20 degrees C and dual sharp electrode recordings at 35 degrees C. In each group, a range of morphological and electrophysiological characteristics was observed. In all groups, however, positive correlations were found between the width of the action potential and rise times and widths at half-amplitude of EPSPs and IPSPs and the EPSP paired pulse ratio. Multipolar interneurones with narrow spikes generated the fastest IPSPs in pyramidal cells and received the briefest, most strongly depressing EPSPs, while bitufted interneurones with broader spikes and adapting and burst firing patterns activated the broadest IPSPs and received the slowest, most strongly facilitating/augmenting EPSPs. Correlations were similar in all groups, with no significant differences between adult rat and cat, or between layers, but events were four times slower in juveniles at 20 degrees C. Comparisons with previous studies indicate that this is due in part to age, but in large part to temperature. Studies in adults were extended with detailed analysis of synaptic dynamics, which appeared to decay more rapidly than at juvenile connections. EPSPs exhibited the complexity in time course of facilitation, augmentation and depression previously described in other adult neocortical connections. That is, the time course of recovery from facilitation or depression rarely followed a simple smooth exponential decay. Facilitation and depression were not always maximal at the shortest interspike intervals, and recovery was often interrupted by peaks and troughs in mean EPSP amplitude with a periodicity around 80 Hz.

Electrical coupling between pyramidal cells in adult cortical regions.

Recently, intense interest has focussed on electrical coupling between interneurones in cortical regions and their contributions towards oscillatory network activity. Despite mounting circumstantial evidence that pyramidal cells are also coupled, the paucity of direct evidence has made this controversial. Dual intracellular recordings from pairs of cortical and hippocampal pyramids demonstrated strong, but sparse coupling. Approximately 70% of CA1 pyramids close to the stratum radiatum border were coupled to another pyramid, but only to one or two of their very closest neighbours. On average 25% of the steady state and 10% of the peak action potential voltage change in one cell transferred to the other, supporting synchrony and promoting burst firing. The very high incidence of convergent inputs from coupled pyramids onto single targets provided additional evidence that 'spikelets' reflected full action potentials in a coupled cell, since the EPSPs activated by APs and by 'spikelets' had significantly different amplitude distributions.

CBP-induced stimulation of c-Fos activity is abrogated by E1A.

The CBP protein stimulates transcription of cAMP-responsive genes by binding to the phosphorylated activation domain of the CREB transcription factor. Here we show that CBP stimulates transcription of Fos/Jun activity in F9 cells and that this response of mediated, at least partly, via c-Fos. We show that CBP binds c-Fos in a phosphorylation-independent manner in vitro, using a domain distinct from that required to bind CREB. When this CBP domain is linked to the activation domain of VP16 it can stimulate GAL4-Fos activity in vivo. The domain of CBP that binds c-Fos is also used to contact the E1A protein. We therefore asked whether the documented repression of AP1 activity by E1A is due to sequestration of CBP from c-Fos. We show that E1A 12S can repress c-Fos activation functions. The use of E1A mutants indicates that binding of CBP, but not RB, to E1A is essential for E1A-mediated repression. These data support a model whereby E1A can modulate AP1 activity by directly competing for the CBP co-activator protein.

Interlaminar connections in the neocortex.

This review summarizes the local circuit, interlaminar connections in adult mammalian neocortex. These were first demonstrated with anatomical techniques, which indicate some of the exquisite spatial precision present in the circuitry. Details, such as the class(es) of neurons targeted by some of these projections, have begun to be added in studies that combine paired/triple intracellular recordings with dye-filling of connected neurons. Clear patterns are emerging from these studies, with 'forward' projections from layer 4 to 3 and from 3 to 5 targeting both selected pyramidal cells and interneurons, while 'back' projections from layer 5 to 3 and from 3 to 4 target only interneurons. To place these data in a wider context, the major afferent inputs to and efferent outputs from each of the layers are discussed first.

The CBP co-activator is a histone acetyltransferase.

The CBP protein acts as a transcriptional adaptor for many different transcription factors by directly contacting DNA-bound activators. One mechanism by which CBP is thought to stimulate transcription is by recruiting the histone acetyltransferase (HAT) P/CAF to the promoter. Here we show that CBP has intrinsic HAT activity. The HAT domain of CBP is adjacent to the binding site for the transcriptional activator E1A. Although E1A displaces P/CAF from CBP, it does not disrupt the CBP-associated HAT activity. Thus E1A carries HAT activity when complexed with CBP. Targeting CBP-associated HAT activity to specific promoters may therefore be a mechanism by which E1A acts as a transcriptional activator.

Regulation of gene expression by transcription factor acetylation.

In the nucleus, DNA is tightly packaged into higher-order structures, generating an environment that is highly repressive towards DNA processes such as gene transcription. Acetylation of lysine residues within proteins has recently emerged as a major mechanism used by the cell to overcome this repression. Acetylation of non-histone proteins, including transcription factors, as well as histones, appears to be involved in this process. Like phosphorylation, acetylation is a dynamic process that can regulate protein-DNA and protein-protein interactions. Moreover, a conserved domain, the bromodomain, has been implicated in the binding of acetylated peptides, suggesting a role for acetylation in intracellular signalling.