Proximal P2 Research Articles

RUNX1 isoforms regulate RUNX1 and target genes differentially in platelets-megakaryocytes: association with clinical cardiovascular events

BackgroundHematopoietic transcription factor RUNX1 is expressed from proximal P2 and distal P1 promoters to yield isoforms RUNX1 B and C, respectively. The roles of these isoforms in RUNX1 autoregulation and downstream gene regulation in megakaryocytes and platelets are unknown. ObjectivesTo understand the regulation of RUNX1 and its target genes by RUNX1 isoforms. MethodsWe performed studies on RUNX1 isoforms in megakaryocytic human erythroleukemia (HEL) cells and HeLa cells (lack endogenous RUNX1), in platelets from 85 healthy volunteers administered aspirin or ticagrelor, and on the association of RUNX1 target genes with acute events in 587 patients with cardiovascular disease (CVD). ResultsIn chromatin immunoprecipitation and luciferase promoter assays, RUNX1 isoforms B and C bound and regulated P1 and P2 promoters. In HeLa cells, RUNX1B decreased and RUNX1C increased P1 and P2 activities, respectively. In HEL cells, RUNX1B overexpression decreased RUNX1C and RUNX1A expression; RUNX1C increased RUNX1B and RUNX1A. RUNX1B and RUNX1C regulated target genes (MYL9, F13A1, PCTP, PDE5A, and others) differentially in HEL cells. In platelets, RUNX1B transcripts (by RNA sequencing) correlated negatively with RUNX1C and RUNX1A; RUNX1C correlated positively with RUNX1A. RUNX1B correlated positively with F13A1, PCTP, PDE5A, RAB1B, and others, and negatively with MYL9. In our previous studies, RUNX1C transcripts in whole blood were protective against acute events in CVD patients. We found that higher expression of RUNX1 targets F13A1 and RAB31 associated with acute events. ConclusionRUNX1 isoforms B and C autoregulate RUNX1 and regulate downstream genes in a differential manner, and this is associated with acute events in CVD.

RUNX1 Isoforms Regulate RUNX1 and Target-Genes Differentially in Platelets-Megakaryocytes: Association with Clinical Cardiovascular Events.

Hematopoietic transcription factor RUNX1 is expressed from proximal P2 and distal P1 promoter to yield isoforms RUNX1 B and C, respectively. The roles of these isoforms in RUNX1 autoregulation and downstream-gene regulation in megakaryocytes and platelets are unknown. To understand the regulation of RUNX1 and its target genes by RUNX1 isoforms. We performed studies on RUNX1 isoforms in megakaryocytic HEL cells and HeLa cells (lack endogenous RUNX1), in platelets from 85 healthy volunteers administered aspirin or ticagrelor, and on the association of RUNX1 target genes with acute events in 587 patients with cardiovascular disease (CVD). In chromatin immunoprecipitation and luciferase promoter assays, RUNX1 isoforms B and C bound and regulated P1 and P2 promoters. In HeLa cells RUNX1B decreased and RUNX1C increased P1 and P2 activities, respectively. In HEL cells, RUNX1B overexpression decreased RUNX1C and RUNX1A expression; RUNX1C increased RUNX1B and RUNX1A. RUNX1B and RUNX1C regulated target genes (MYL9, F13A1, PCTP, PDE5A and others) differentially in HEL cells. In platelets RUNX1B transcripts (by RNAseq) correlated negatively with RUNX1C and RUNX1A; RUNX1C correlated positively with RUNX1A. RUNX1B correlated positively with F13A1, PCTP, PDE5A, RAB1B, and others, and negatively with MYL9. In our previous studies, RUNX1C transcripts in whole blood were protective against acute events in CVD patients. We found that higher expression of RUNX1 targets F13A1 and RAB31 associated with acute events. RUNX1 isoforms B and C autoregulate RUNX1 and regulate downstream genes in a differential manner and this associates with acute events in CVD.

Complete fractures through osseous cyst‐like lesions of the proximal phalanx in three horses

SummaryThis case series describes three cases of osseous cyst‐like lesions of the proximal phalanx (P1), which were associated with a complete fracture. The fractures and osseous cyst‐like lesions were analysed through radiographic and computed tomographic (CT) images for fracture configuration and prodromal factors, such as subchondral sclerosis and periosteal new bone formation. Internal stabilisation was performed in all cases through placing transcortical bone screws under CT guidance. In conclusion, the presence of osseous cyst‐like lesions in the proximal P1 as well as the addition of prodromal markers, such as localised periosteal new bone formation and subchondral bone sclerosis, suggest pathological subchondral bone with the potential to fracture and stabilisation should be prioritised.

RUNX1 Isoforms Regulate RUNX1 and Target-Genes Differentially in Platelets/Megakaryocytes: Association with Clinical Cardiovascular Events

RUNX1 is an essential transcription factor for hematopoiesis. Germline RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and predisposition to myeloid malignancies. Three major RUNX1 isoforms (A, B and C) are recognized and share a DNA binding RUNT domain; RUNX1A lacks the transactivation domain. Two distinct promoters regulate RUNX1 expression: distal P1 regulates RUNX1C; proximal P2 regulates RUNX1B and RUNX1A. Little is known regarding the differential effects of isoforms in RUNX1 autoregulation, target-gene regulation and association with clinical events. We performed studies in megakaryocytic HEL cells, HeLa cells (which do not express RUNX1) and in platelets of healthy subjects (n=74) and provide evidence for isoform-specific differential autoregulation and target-gene regulation by RUNX1. We show an association between expression of specific RUNX1-regulated genes in whole blood with cardiovascular events in patients. There are 5 RUNX1 consensus binding sites in P1 promoter and 1 in P2 promoter within ~1000 bp from ATG. Wild type RUNX1 P1 and P2 promoter regions were individually cloned into PGL4 luciferase promoter vector. ChIP studies using PMA-treated megakaryocytic HEL cells showed RUNX1 binding to chromatin regions encompassing the RUNX1 binding sites in both promoters. Mutations of 2 RUNX1 binding sites in intron region of P1 promoter reduced promoter activity; mutations of first 2 RUNX1 binding sites at the exon region of P1 promoter increased activity. Mutation of single RUNX1 binding site in P2 promoter increased promoter activity. Thus, RUNX1 binds to P1 and P2 promoters to regulate activities. To examine the RUNX1 autoregulation by individual isoforms, we co-transfected each isoform with P1 or P2 promoter vector in HeLa cells, which do not express endogenous RUNX1. In response to RUNX1B over-expression, both P1 and P2 promoters showed a dose-dependent decrease in promoter activity. RUNX1C over-expression increased P1 and P2 promoter activities. Thus, RUNX1B and RUNX1C regulate P1 and P2 promoters differentially. In HEL cells, which have endogenous RUNX1, RUNX1B overexpression decreased RUNX1C and RUNX1A mRNA/protein expression by ~50%. RUNX1C overexpression increased RUNX1B and RUNX1A mRNA/protein expression. Thus, RUNX1B decreases RUNX1C and RUNX1A expression; RUNX1C increases RUNX1B and RUNX1A. We studied the regulation by RUNX1 isoforms of target genes PCTP (phosphatidylcholine transfer protein), MYL9 (myosin light chain), PDE5A (phosphodiesterase 5A) and F13A (factor XIIIA) . In HeLa cells. RUNX1B overexpression increased PCTP, MYL9 and PDE5A (protein and mRNA); RUNX1C overexpression reduced PCTP and MYL9. In HEL cells RUNX1B increased PCTP, MYL9, PDE5A and F13A expression; RUNX1C reduced PCTP and MYL9 expression. To obtain evidence in vivo, we studied the relationships between RUNX1 isoforms and their relationships to RUNX1-target genes by RNAseq in leukocyte-poor platelets (74 healthy donors; ages 30-75 yrs). RUNX1B correlated negatively with RUNX1C (R= -0.413 and p=1.5e-06) and RUNX1A (R= -0.493 and p=0.00e+oo); RUNX1C correlated positively with RUNX1A (R=0.45, p<0.0001). RUNX1B correlated positively with PCTP, F131A, PDE5A and RAB1B, and negatively with MYL9 (Figure 1); no significant relationships were observed with RUNX1C or RUNX1A. To understand the relationships to cardiovascular events (MI or death), we explored whole blood gene expression using microarrays in 587 patients (2 cohorts, 190 and 397 patients) presenting to cardiac catherization for chest pain and followed for 3.8 years. In our previous studies RUNX1B transcripts were higher and RUNX1C lower in those with MI or death. We now found that higher expressions of F13A1(Figure 2) and RAB31, which have been shown by us to be RUNX1-targets, were associated with cardiovascular events. Conclusions: RUNX1 isoforms autoregulate RUNX1 expression differentially and regulate target genes in a differential manner, a pattern consistent with in vivo platelet gene expression. The relative expression of RUNX1B and RUNX1C isoforms and of their target genes associate with clinical cardiovascular events. RUNX1-isoform specific effects need to be considered in clinical scenarios where RUNX1 is involved and in its pharmacologic modulation.

Translated article] Comparison of three different minimally invasive intramedullary screw for proximal phalanx fractures. A biomechanical study

Background and objectivePhalangeal fractures are the most common hand fractures. In the last years, intramedullary compression screw (IMHCS) for instable transverse or short oblique proximal P1 fractures have been described. Although both anterograde (intraarticular or trans-articular) and retrograde IMHCS techniques have shown good results, no comparison between anterograde and retrograde screw in P1 fractures has been published. We sought to determine stability with retrograde IMHCS and anterograde IMHCS, both trans-articular and intra-articular technique, in a cadaveric transverse proximal P1 fracture model, at two different levels. Material and methodsWe performed a biomechanical study in 30 fresh-frozen human cadaveric P1 fracture model. Fracture was performed at 9-mm from the metacarpo-phalangeal (MCP) joint in 15 specimens, whereas it was done at 15mm in the other 15. In turn, in each group, five fractures were stabilised with an anterograde intra-articular IMHCS, five with anterograde trans-articular IMHCS and other five with retrograde IMHCS. ResultsAnterograde IMHCS fixation in 9-mm P1 fractures (both trans- and intra-articular technique, 62.74N and 70.86N, respectively) was found to be more stable than retrograde IMHCS one (32.72N) (p=0.022). Otherwise, retrograde IMHCS fixation was found to be more stable in more distal P1 fractures (90.52N retrograde vs. 57.64N trans-articular vs. 42.92N intra-articular; p=0.20). ConclusionsAnterograde IMHCS fixation in proximal transverse P1 bone cut in a cadaveric model provides more stability than retrograde IMHCS, while retrograde screw provides more stability when the bone cut is located more distal.

Comparación de tres técnicas de tornillo endomedular mínimamente invasivo para fracturas de falange proximal. Estudio biomecánico

Background and objectivePhalangeal fractures are the most common hand fractures. In the last years, intramedullary compression screw (IMHCS) for instable transverse or short oblique proximal P1 fractures have been described. Although both anterograde (intraarticular or trans-articular) and retrograde IMHCS techniques have shown good results, no comparison between anterograde and retrograde screw in P1 fractures has been published. We sought to determine stability with retrograde IMHCS and anterograde IMHCS, both trans-articular and intra-articular technique, in a cadaveric transverse proximal P1 fracture model, at two different levels. Material and methodsWe performed a biomechanical study in 30 fresh-frozen human cadaveric P1 fracture model. Fracture was performed at 9-mm from the metacarpo-phalangeal (MCP) joint in 15 specimens, whereas it was done at 15 mm in the other 15. In turn, in each group, five fractures were stabilized with an anterograde intra-articular IMHCS, five with anterograde trans-articular IMHCS and other five with retrograde IMHCS. ResultsAnterograde IMHCS fixation in 9-mm P1 fractures (both trans- and intra-articular technique, 62.74 N and 70.86 N, respectively) was found to be more stable than retrograde IMHCS one (32.72 N) (p = 0.022). Otherwise, retrograde IMHCS fixation was found to be more stable in more distal P1 fractures (90.52 N retrograde vs. 57.64 N trans-articular vs. 42.92 N intra-articular; p = 0.20). ConclusionsAnterograde IMHCS fixation in proximal transverse P1 bone cut in a cadaveric model provides more stability than retrograde IMHCS, while retrograde screw provides more stability when the bone cut is located more distal.

Microsurgical treatment of posterior inferior cerebellar aneurysms based on angioarchitecture supplemented by high-resolution vessel wall MRI: a case series report

BackgroundSurgical treatment of posterior inferior cerebellar artery (PICA) aneurysms is challenging because many are nonsaccular and atherosclerotic. We report our tailored approach to PICA aneurysms, which is based on angioarchitecture...

Trapping and P2-P3 Posterior Inferior Cerebellar Artery Reanastomosis for Treatment of a Ruptured Fusiform Aneurysm: Application of Fourth-Generation Technique: 2-Dimensional Operative Video.

Aneurysms of the posterior inferior cerebellar artery (PICA) represent the second most common posterior circulation aneurysm and commonly have complex morphology. Various bypass options exist for PICA aneurysms,1-6 depending on their location relative to brainstem perforators and the vertebral artery, and the presence of nearby donor arteries. We present a case of a man in his late 40s who presented with 3 d of severe headache. He was found to have a fusiform right P2-segment PICA aneurysm. Preoperative angiogram demonstrated the aneurysm and a redundant P3 caudal loop that came in close proximity to the healthy P2 segment proximal to the aneurysm. The risks and benefits of the procedure were discussed with the patient, and they consented for a right far lateral approach craniotomy with partial condylectomy for trapping of the aneurysm with bypass. The aneurysm was trapped proximally and distally. The P3 was transected just distal to the aneurysm and brought toward the proximal P2 segment, facilitated by a lack of perforators on this redundant distal artery. An end-to-side anastomosis was performed. Postoperative angiogram demonstrated exclusion of the aneurysm and patent bypass. The patient recovered well and remained without any neurological deficit at 6-mo follow-up. This case demonstrates the use of a "fourth-generation"5,7,8 bypass technique. These techniques represent the next innovation beyond third-generation intracranial-intracranial bypass. In this type 4B reanastomosis bypass, an unconventional orientation of the arteries was used. Whereas reanastomosis is typically performed end-to-end, the natural course of these arteries and the relatively less-mobile proximal P2 segment made end-to-side the preferred option in this case. Fourth-generation bypass techniques open up more configurations for reanastomosis, using the local anatomy to the surgeon's advantage. The patient consented to the described procedure and consented to the publication of their image.

From the Editor’s desk…

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Novel function of SART1 in HNF4α transcriptional regulation contributes to its antiviral role during HBV infection

Our understanding of the interactions between HBV and its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict HCV. Thus, we aimed to dissect its role in HBV infection. SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV-infected cell cultures and invivo in HBV-infected mice. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) was used to investigate protein-DNA interactions. In HBV-infected cell cultures, downregulation of SART1 did not affect covalently closed circular HBV DNA but resulted in markedly enhanced HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV-infected mice persistently replicating HBV. Inhibition of Janus kinases had no effect on SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with the HNF4α proximal P1 promoter element. We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon-stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression, which is essential for transcription of HBV cccDNA. Hepatitis B virus (HBV) infects hepatocytes and persists in the form of covalently closed circular DNA (cccDNA), which remains a major obstacle to successful antiviral treatment. In this study, using various HBV models, we demonstrate that the protein SART1 restricts HBV cccDNA transcription by suppressing a key transcription factor, HNF4α.

Targeted Therapy for AML Expressing RUNX1 Mutation

RUNX1 is the DNA-binding subunit of the core binding factor (CBF) complex and a transcription factor (TF) involved in normal and malignant hematopoiesis. Transcriptionally active full-length RUNX1b isoform is transcribed from 6 exons, encoding a DNA binding ‘Runt’ homology domain and a more C-terminal transcription activation domain. Within its CTCF-anchored loop or TAD (topologically-associating domain), RUNX1 gene has a highly conserved +24-kb enhancer (eR1 in mouse), which is part of a large super-enhancer (>170 kb) covering the entire intron 1 between proximal P2 and distal P1 promoter of RUNX1 and controlling the transcription of RUNX1. TFs bind to super-enhancers/enhancers that loop over to interact with promoters to transcriptionally regulate oncogenes, such as RUNX1 and its targets, as well as multiple other AML-relevant oncogenes involved in growth and survival of AML blast progenitor cells (BPCs). Heatmap of Hi-C interaction scores within the RUNX1 TAD showed triangle shaped regions of high interaction scores highlighting interactions between +24-kb enhancer and the P2 promoter of RUNX1. To functionally validate these findings, we cloned and transfected into HEK293 cells expressing RUNX1, a luciferase reporter construct containing the DNA of the P2 promoter with or without the +24 kb-enhancer of RUNX1. Our findings confirm that, as compared to the promoter or the enhancer alone, the +24 kb-enhancer-driven P2 promoter exhibited superior luciferase induction representing increased transcriptional activity of RUNX1. Somatic mutations in RUNX1 commonly occur in MDS, as well as in the secondary (s) or de novo AML (~10%). Majority of mutant (mt) RUNX1 are missense, large deletions or truncation-mutations, behaving mostly as loss of function mutations. Presence of mtRUNX1 confers relative therapy-resistance and poorer survival in patients with AML. Lack of specific targeted therapy coupled with resistance to standard chemotherapy may account for the poorer outcome in AML expressing somatic or germline mtRUNX1. Therefore, there is a strong rationale and an unmet need to develop novel targeted therapies for AML expressing somatic or germline mtRUNX1. In the present studies, we demonstrate for the first time that shRNA mediated knockdown of RUNX1 expression inhibits growth and induces differentiation and apoptosis of cultured cell line (OCI-AML5) and patient-derived (PD) AML BPCs expressing mtRUNX1. After engraftment, doxycycline-inducible shRNA-mediated in vivo knockdown of RUNX1 in OCI-AML5 cells, compared to controls, markedly reduced AML burden and improved survival of the immune-depleted (NSG) mice engrafted with luciferase transduced OCI-AML5 cells. Interaction of TFs with DNA and transcriptional co-factors such as the histone acetyltransferase p300 at enhancers and promoters leads to acetylation of histone proteins and TFs. BET (Bromodomain Extra-terminal) protein (BETP) BRD4 binds to acetylated histones and TFs at super-enhancers and recruits transcriptional co-factors to promote RNA pol II (RNAP2)-mediated transcription of oncogenes and their targets. ChIP-Seq analysis with anti-BRD4 and anti-H3K27Ac antibody showed that Runx1 super-enhancer and P2 promoter are occupied by BRD4. shRNA mediated knockdown of BRD4 attenuated expression of RUNX1 and its targets, MYC, PU.1 and myeloperoxidase (MPO), inhibited growth and induced apoptosis of OCI-AML5 cells. We also found that BETP-PROTACs (proteolysis targeted chimera) ARV-825 and its pharmacologically superior version ARV-771 (20 to 250 nM) caused degradation and depletion of BRD4, reduced BRD4 occupancy at the+24-kb-enhancer and P2 promoter of RUNX1, repressing RUNX1 and its targets, and potently induced apoptosis of OCI-AML5, Mono-Mac-1 and PD AML BPCs expressing mtRUNX1. Treatment with ARV-771 (30 mg/kg SQ daily for 3 weeks also inhibited AML growth and improved survival of NSG mice engrafted with luciferase-transduced OCI-AML5 cells. By utilizing the RNA-Seq mRNA signature from RUNX1-depleted AML cells we queried for expression mimickers (EMs) through LINCS1000-CMap (Connectivity Mapping) analyses. This uncovered several novel agents that inhibit RUNX1 expression and induce in vitro lethality and reduce in vivo leukemia burden in AML BPCs expressing mtRUNX1. These findings highlight RUNX1-targeted novel agents to be developed and tested as potential therapy for AML expressing mtRUNX1. DisclosuresDiNardo:AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding. Qian:4Arvinas, LLC. New Haven, CT: Employment. Raina:4Arvinas, LLC. New Haven, CT: Employment. Takahashi:Symbio Pharmaceuticals: Consultancy.

How does bone strain vary between the third metacarpal and the proximal phalangeal bones of the equine distal limb?

Strain parameters at injury prone sites of the equine third metacarpal (MC3) and proximal phalangeal (P1) bones were investigated with the aim of improving understanding of injury pathogenesis. We hypothesized that dorsal principal and shear strain patterns, magnitudes and directions would differ from proximal-to-distal; and would be similar from medial-to-lateral across each bone. Unilateral limbs from nine equine cadavers were instrumented with rosette strain gauges during limb loading to 10,500 N. Gauges were attached at seven dorsal sites: middle MC3, distal MC3 (medial, middle, lateral) and proximal P1 (medial, middle, lateral). Outcome measures were analysed with repeated measures analysis of variance. Distal MC3 had the greatest, and proximal P1 the smallest magnitude of minimum principal and shear strains. Directions of maximum and minimum principal strain were similar at the middle and distal MC3 sites with a 20-40° direction difference compared to proximal P1. The patterns of strain magnitude and direction were similar from medial-to-lateral on distal MC3 but varied in pattern and magnitude among the P1 sites. Overall, as load reached maximum, direction of minimum principal strain became more axial in orientation, converging from opposite directions between bones, potentially maximising stability of the distal limb. The difference in strain parameters and strain ratio for adjacent anatomic sites on distal MC3 and proximal P1 was not anticipated, in light of the anatomic congruity of the metacarpophalangeal joint. Based on the predominance of shear strain across proximal P1, shear forces are likely the predominant biomechanical contributor to sagittal fractures of P1.

Ex vivo comparison of standing and recumbent repair of incomplete parasagittal fractures of the first phalanx in horses.

To assess suspensory ligament extensor branch location and fracture gap reduction with simulation of standing and recumbent cortical bone screw repair of experimental incomplete parasagittal proximal phalanx (P1) fractures. Controlled laboratory study. Twenty equine cadaver forelimbs. Simulated fractures were repaired twice in random order. A proximal cortical bone screw was placed in lag fashion with the limb unloaded (simulated recumbent repair) and loaded to 38% of body weight (range, 375-568 kg; simulated standing repair). Changes in fracture gap width were assessed on computed tomography (CT) images and with intraplanar force-sensitive resistors measuring voltage ratios (V△ ) between loaded recumbent (R-1) and standing repair simulations (R-2). Extensor branch borders were determined relative to implant position and sagittal P1 width on transverse CT images. P ≤ .05 was considered significant. Standing repair simulation-associated fracture gaps were not wider than in R-1 while controlling for confounding factors (loading weight, implant position, or animal age; P > .7, repeated-measures analysis of variance). Voltage ratio data associated with R-2 were not smaller than with R-1 (mean difference, 0.002 ± 0.052; one-sided Wilcoxon signed-rank test, P = .27). More of P1 width was approachable palmar to extensor branches when limbs were loaded (0.804 ± 0.314 cm) vs unloaded (0.651 ± 0.31 cm; paired Student's t test, P < .001). Simulated standing repair was not associated with inferior fracture reduction compared with loaded simulations of recumbent repairs. Limb loading affected extensor branch location relevant to implant positioning. Unloading during standing repair of incomplete parasagittal proximal P1 fractures may not be required to optimize fracture reduction.

Structural insights into the multivalent binding of the Arabidopsis FLOWERING LOCUS T promoter by the CO-NF-Y master transcription factor complex.

Flowering plants sense various environmental and endogenous signals to trigger the floral transition and start the reproductive growth cycle. CONSTANS (CO) is a master transcription factor in the photoperiod floral pathway that integrates upstream signals and activates the florigen gene FLOWERING LOCUS T (FT). Here, we performed comprehensive structural and biochemical analyses to study the molecular mechanism underlying the regulation of FT by CO in Arabidopsis thaliana. We show that the four previously characterized cis-elements in the FT promoter proximal region, CORE1, CORE2, P1, and P2, are all direct CO binding sites. Structural analysis of CO in complex with NUCLEAR FACTOR-YB/YC (NF-YB/YC) and the CORE2 or CORE1 elements revealed the molecular basis for the specific recognition of the shared TGTG motifs. Biochemical analysis suggested that CO might form a homomultimeric assembly via its N-terminal B-Box domain and simultaneously occupy multiple cis-elements within the FT promoter. We suggest that this multivalent binding gives the CO-NF-Y complex high affinity and specificity for FT promoter binding. Overall, our data provide a detailed molecular model for the regulation of FT by the master transcription factor complex CO-NF-Y during the floral transition.

Acute bilateral paramedian thalamic and mesencephalic infarcts due to occlusion of the Percheron artery: a case report

The thalamus and midbrain mesencephalon have a complex blood supply with a great number of feeding arteries. The blood build up of thalamus is being provided by four arteries originating from the vertebrobasilar system and posterior communicating artery. The paramedian thalamic artery is derived from the posterior cerebral artery proximal P1 segment. The infarcts of arterial constitute approximately 35% of all thalamic infarcts. However, bilateral infarctions of this region are very infrequent. Percheron artery originates from the first branch of the posterior cerebral artery and gives bilateral medial thalamic perforating branches. We described a patient of a 88-year-old woman with acute bilateral thalamic and midbrain infarcts due to occlusion of Percheron artery demonstrated on magnetic resonance imaging.

Targeted Disruption of the Cohesin Subunit STAG2 Leads to Loss of Insulation and Inappropriate Gene Activation in Response to Differentiation Signals

Cohesin is a multiprotein complex that mediates sister chromatid cohesion during S phase of the cell cycle, but it also plays crucial roles in genome organization and transcription in interphase. Cohesin's role in regulation of the genome helps determine lineage identity and differentiation of hematopoietic cells (Viny et al., JEM, 2015; Mullenders et al., JEM, 2015; Mazumdar et al., Cell stem cell, 2015; Galeev et al., Cell reports, 2016). Mitotic cohesin is composed of RAD21, SMC1A, SMC3 and STAG1/STAG2 subunits. Mutations in the subunits of cohesin complex are present in significant proportion of myeloid malignancies (Thota et al., Blood 2014). In particular, the frequency of cohesin subunit mutation is as high as ~50% in Down-syndrome associated megakaryoblastic leukemia subtype (Yoshida et al., Nature Genetics, 2013).To further understand cohesin's role in gene regulation in a leukemia context, we used CRISPR-CAS9 to engineer a leukemia associated STAG2 mutation, R614*, into K562 leukemia cells to generate isogenic model lines. Homozygous mutant K562 cells showed complete loss of STAG2 expression. We also obtained STAG2 R614* heterozygous cell clones that showed partial loss of STAG2 expression (STAG2 is on the X chromosome, and the wild type allele is mosiacally silenced in heterozygotes).Phenotype analyses of STAG2 mutant K562 cells showed that homozygous mutants had altered morphology and had acquired adherent properties. Homozygous mutant cells activated expression of the stem cell marker, KIT, and showed a reduction in the neutrophil differentiation marker, CD15.We used ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to examine changes in chromatin accessibility, and RNA sequencing to determine changes in gene expression in STAG2 mutants. More than 5,000 genes were dysregulated in STAG2 homozygous mutants, accompanied by profound changes in chromatin accessibility with gain and loss of accessibility represented equally. Loss of accessibility was observed at insulators that recruit CCCTC-binding factor (CTCF), indicating potential loss of insulation at chromatin boundaries. Conversely, gain of chromatin accessibility was observed at regions that are normally repressed in K562, and this was accompanied by activation of transcription factors such as ERG and FLI1. Gain of accessibility was also observed at myeloid-associated super enhancers of the RUNX1 gene.We hypothesized that loss of STAG2 leads to an inability to constrain transcriptional programs upon differentiation of leukemia cells. To test this hypothesis, we stimulated the STAG2 mutant and parental K562 cells with phorbol myristate acetate (PMA) to induce megakaryocyte differentiation. PMA-stimulated STAG2 homozygous mutant cells showed a striking precocious spike in RUNX1 expression that was associated with enhanced transcription from its proximal P2 promoter. A similar precocious spike was observed in transcription of ERG. Interestingly, the spike in transcription of these genes was not observed at 48 hours, indicating that the ectopic transcription of RUNX1 and ERG is confined to early response to stimulation. The BET inhibitor JQ1, was previously shown to block enhancer activity (Bhagwat et al., Cell reports, 2016). Treatment of STAG2 mutant cells with JQ1 dampened ectopic RUNX1 P2 expression and led to a complete loss of RUNX1 P1 and ERG activation during PMA stimulation. These results suggest that ectopic RUNX1 expression in response to STAG2 deficiency is enhancer-driven. Furthermore, JQ1 treatment caused a 2-fold decrease in KIT levels in STAG2-deficient cells, indicating a reversal of immature properties in these cells.In summary, our results suggest that STAG2 mutation results in loss of chromatin insulation, leading to inappropriate activation of hematopoietic enhancers. Loss of constraints on enhancer-driven transcription could in turn underlie the aberrant gene expression in STAG2-deficient cells exposed to differentiation cues. DisclosuresNo relevant conflicts of interest to declare.

Intestine-specific expression of human chimeric intestinal alkaline phosphatase attenuates Western diet-induced barrier dysfunction and glucose intolerance.

Intestinal epithelial cell derived alkaline phosphatase (IAP) dephosphorylates/detoxifies bacterial endotoxin lipopolysaccharide (LPS) in the gut lumen. We have earlier demonstrated that consumption of high‐fat high‐cholesterol containing western type‐diet (WD) significantly reduces IAP activity, increases intestinal permeability leading to increased plasma levels of LPS and glucose intolerance. Furthermore, oral supplementation with curcumin that increased IAP activity improved intestinal barrier function as well as glucose tolerance. To directly test the hypothesis that targeted increase in IAP would protect against WD‐induced metabolic consequences, we developed intestine‐specific IAP transgenic mice where expression of human chimeric IAP is under the control of intestine‐specific villin promoter. This chimeric human IAP contains domains from human IAP and human placental alkaline phosphatase, has a higher turnover number, narrower substrate specificity, and selectivity for bacterial LPS. Chimeric IAP was specifically and uniformly overexpressed in these IAP transgenic (IAPTg) mice along the entire length of the intestine. While IAP activity reduced from proximal P1 segment to distal P9 segment in wild‐type (WT) mice, this activity was maintained in the IAPTg mice. Dietary challenge with WD impaired glucose tolerance in WT mice and this intolerance was attenuated in IAPTg mice. Significant decrease in fecal zonulin, a marker for intestinal barrier dysfunction, in WD fed IAPTg mice and a corresponding decrease in translocation of orally administered nonabsorbable 4 kDa FITC dextran to plasma suggests that IAP overexpression improves intestinal barrier function. Thus, targeted increase in IAP activity represents a novel strategy to improve WD‐induced intestinal barrier dysfunction and glucose intolerance.

Ileum-Specific Transgenic Expression of Human Chimeric Intestinal Alkaline Phosphatase (IAP) Attenuates Western Diet (WD)–Induced Barrier Dysfunction and Glucose Intolerance

Intestinal epithelial cell derived IAP dephosphorylates/detoxifies bacterial endotoxin LPS in the gut lumen. We have earlier demonstrated that improvement of intestinal barrier function was accompanied by an increase in IAP activity. Herein, we developed IAP transgenic mice where expression of human chimeric IAP is under the control of intestine specific villin promoter. As shown in Panel A, compared to non-transgenic wild type (WT) littermates, IAP expression (identified by c-myc tag) was noted in colon, ileum jejunum and duodenum of IAP-transgenic (IAPTg) mice. IAP was almost uniformly expressed along the length of the ileum (Panel B) and while IAP activity reduced from proximal P1 segment to distal P9 segment in WT mice, this activity was maintained in the IAPTg mice (Panel C). Dietary challenge with WD impaired glucose tolerance in WT mice and this intolerance was attenuated in IAPTg mice (Panels D and E). Significant decrease in fecal zonulin, a marker for intestinal barrier dysfunction, in WD fed IAPTg mice (Panel F) and a corresponding decrease in translocation of oral 4kDa FITC dextran to plasma (Panel G) suggests that IAP overexpression improves intestinal barrier function. Thus, targeted increase in IAP activity represents a novel strategy to improve WD induced intestinal dysfunction and glucose intolerance. Disclosure H. He: None. S.S. Ghosh: None. P.J. Yannie: None. J. Wang: None. S. Ghosh: None.

Mouse RUNX1C regulates premegakaryocytic/erythroid output and maintains survival of megakaryocyte progenitors

AbstractRUNX1 is crucial for the regulation of megakaryocyte specification, maturation, and thrombopoiesis. Runx1 possesses 2 promoters: the distal P1 and proximal P2 promoters. The major protein isoforms generated by P1 and P2 are RUNX1C and RUNX1B, respectively, which differ solely in their N-terminal amino acid sequences. RUNX1C is the most abundantly expressed isoform in adult hematopoiesis, present in all RUNX1-expressing populations, including the cKit+ hematopoietic stem and progenitor cells. RUNX1B expression is more restricted, being highly expressed in the megakaryocyte lineage but downregulated during erythropoiesis. We generated a Runx1 P1 knock-in of RUNX1B, termed P1-MRIPV. This mouse line lacks RUNX1C expression but has normal total RUNX1 levels, solely comprising RUNX1B. Using this mouse line, we establish a specific requirement for the P1-RUNX1C isoform in megakaryopoiesis, which cannot be entirely compensated for by RUNX1B overexpression. P1 knock-in megakaryocyte progenitors have reduced proliferative capacity and undergo increased cell death, resulting in thrombocytopenia. P1 knock-in premegakaryocyte/erythroid progenitors demonstrate an erythroid-specification bias, evident from increased erythroid colony-forming ability and decreased megakaryocyte output. At a transcriptional level, multiple erythroid-specific genes are upregulated and megakaryocyte-specific transcripts are downregulated. In addition, proapoptotic pathways are activated in P1 knock-in premegakaryocyte/erythroid progenitors, presumably accounting for the increased cell death in the megakaryocyte progenitor compartment. Unlike in the conditional adult Runx1 null models, megakaryocytic maturation is not affected in the P1 knock-in mice, suggesting that RUNX1B can regulate endomitosis and thrombopoiesis. Therefore, despite the high degree of structural similarity, RUNX1B and RUNX1C isoforms have distinct and specific roles in adult megakaryopoiesis.

Transcription Factor RUNX1 Regulates Platelet PCTP (Phosphatidylcholine Transfer Protein): Implications for Cardiovascular Events: Differential Effects of RUNX1 Variants.

PCTP (phosphatidylcholine transfer protein) regulates the intermembrane transfer of phosphatidylcholine. Higher platelet PCTP expression is associated with increased platelet responses on activation of protease-activated receptor 4 thrombin receptors noted in black subjects compared with white subjects. Little is known about the regulation of platelet PCTP. Haplodeficiency of RUNX1, a major hematopoietic transcription factor, is associated with thrombocytopenia and impaired platelet responses on activation. Platelet expression profiling of a patient with a RUNX1 loss-of-function mutation revealed a 10-fold downregulation of the PCTP gene compared with healthy controls. We pursued the hypothesis that PCTP is regulated by RUNX1 and that PCTP expression is correlated with cardiovascular events. We studied RUNX1 binding to the PCTP promoter using DNA-protein binding studies and human erythroleukemia cells and promoter activity using luciferase reporter studies. We assessed the relationship between RUNX1 and PCTP in peripheral blood RNA and PCTP and death or myocardial infarction in 2 separate patient cohorts (587 total patients) with cardiovascular disease. Platelet PCTP protein in the patient was reduced by ≈50%. DNA-protein binding studies showed RUNX1 binding to consensus sites in ≈1 kB of PCTP promoter. PCTP expression was increased with RUNX1 overexpression and reduced with RUNX1 knockdown in human erythroleukemia cells, indicating that PCTP is regulated by RUNX1. Studies in 2 cohorts of patients showed that RUNX1 expression in blood correlated with PCTP gene expression; PCTP expression was higher in black compared with white subjects and was associated with future death/myocardial infarction after adjustment for age, sex, and race (odds ratio, 2.05; 95% confidence interval 1.6-2.7; P<0.0001). RUNX1 expression is known to initiate at 2 alternative promoters, a distal P1 and a proximal P2 promoter. In patient cohorts, there were differential effects of RUNX1 isoforms on PCTP expression with a negative correlation in blood between RUNX1 expressed from the P1 promoter and PCTP expression. PCTP is a direct transcriptional target of RUNX1. PCTP expression is associated with death/myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events.