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1.
Ann Surg Oncol ; 31(2): 974-980, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37973647

ABSTRACT

BACKGROUND: Triple-negative breast cancer (TNBC) is known to portend a worse prognosis compared with same-stage, hormone receptor-positive disease. However, with the recent change in practice to include pembrolizumab in neoadjuvant chemotherapy (NAC) for TNBC, an increase in pathologic complete responses (pCRs) has been reported. The perioperative repercussions of adding pembrolizumab to standard NAC regimens for TNBC are currently unknown. We aimed to explore the perioperative implications of adding pembrolizumab to standard NAC regimens for non-metastatic TNBC. METHODS: This was a retrospective review of the perioperative outcomes in patients with non-metastatic TNBC treated with pembrolizumab-NAC from January 2018 to October 2022 conducted at a high-volume cancer center. Patient demographics, comorbidities, clinical and pathological staging, NAC treatment regimen, initiation, and completion, as well as date of surgery and postoperative complications were analyzed. RESULTS: Of 87 patients, 67.8% had an overall pCR and 86% had an axillary pCR; 37.2% of cN+ patients were spared from axillary lymph node dissection. However, 24.1% of patients experienced surgical complications, 9% of patients were receiving steroids at the time of breast surgery secondary to adverse effects of pembrolizumab-NAC, and 7% underwent a change in the initial surgical plan such as omission of reconstruction. CONCLUSION: Pembrolizumab-NAC has not only significant oncologic benefit but also noteworthy perioperative implications in the surgical management of TNBC.


Subject(s)
Breast Neoplasms , Triple Negative Breast Neoplasms , Humans , Female , Neoadjuvant Therapy , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/surgery , Triple Negative Breast Neoplasms/pathology , Lymphatic Metastasis , Lymph Node Excision , Axilla/pathology
2.
Surg Oncol Clin N Am ; 32(4): 693-703, 2023 10.
Article in English | MEDLINE | ID: mdl-37714637

ABSTRACT

De-escalation of axillary management after neoadjuvant chemotherapy in clinically node-positive patients is feasible. The current literature shows this may be accomplished by sentinel lymph node biopsy (SLNB) with the use of dual tracer and removal of at least 2 sentinel lymph nodes, or by targeted axillary dissection (TAD). The accuracy of TAD has been consistently shown as better than that of SLNB. However, these techniques should only be offered to select patients without extensive axillary disease, understanding that long-term outcomes of minimal axillary surgery in this population are limited at this time.


Subject(s)
Neoadjuvant Therapy , Sentinel Lymph Node , Humans , Lymph Node Excision , Sentinel Lymph Node Biopsy , Axilla
3.
Nat Commun ; 13(1): 7627, 2022 Dec 09.
Article in English | MEDLINE | ID: mdl-36494343

ABSTRACT

DNA methylation is a key epigenetic property that drives gene regulatory programs in development and disease. Current single-cell methods that produce high quality methylomes are expensive and low throughput without the aid of extensive automation. We previously described a proof-of-principle technique that enabled high cell throughput; however, it produced only low-coverage profiles and was a difficult protocol that required custom sequencing primers and recipes and frequently produced libraries with excessive adapter contamination. Here, we describe a greatly improved version that generates high-coverage profiles (~15-fold increase) using a robust protocol that does not require custom sequencing capabilities, includes multiple stopping points, and exhibits minimal adapter contamination. We demonstrate two versions of sciMETv2 on primary human cortex, a high coverage and rapid version, identifying distinct cell types using CH methylation patterns. These datasets are able to be directly integrated with one another as well as with existing snmC-seq2 datasets with little discernible bias. Finally, we demonstrate the ability to determine cell types using CG methylation alone, which is the dominant context for DNA methylation in most cell types other than neurons and the most applicable analysis outside of brain tissue.


Subject(s)
DNA Methylation , High-Throughput Nucleotide Sequencing , Humans , DNA Methylation/genetics , Sequence Analysis, DNA , Epigenomics/methods , Software
4.
Breast Care (Basel) ; 16(3): 276-282, 2021 Jun.
Article in English | MEDLINE | ID: mdl-34248469

ABSTRACT

INTRODUCTION: Breast cancer is the second most common cause of cancer death in females, and 30% of these patients are over the age of 70 years. Studies have shown deviation from the standard treatment paradigms in the elderly, especially in regard to radiation treatment. METHODS: We performed a retrospective chart review on 118 patients over the age of 70 years diagnosed with breast cancer and pathologically proven axillary disease over an 8-year period at an urban academic hospital to examine which patient factors influenced radiotherapy. RESULTS: Increasing patient age was associated with a decrease in the probability of receiving radiotherapy, while HER2-negative patients were more likely to receive radiation. Neither race, number of coexisting medical conditions, or insurance status showed any influence on radiation treatment. CONCLUSION: Patient age has a significant influence if elderly patients with axillary disease receive radiotherapy. Further investigation and validation are needed to understand why chronological age rather than biological age influences treatment modalities.

5.
Am Surg ; 87(1): 120-124, 2021 Jan.
Article in English | MEDLINE | ID: mdl-32845728

ABSTRACT

INTRODUCTION: The 2017 surgical infection society (SIS) guidelines recommend 4 days of antibiotic therapy after source control for complicated intra-abdominal infections (cIAIs). Inappropriate exposure to antibiotics has a negative impact on outcomes in individual patients and populations. The goal of this study was to evaluate our institution's practice patterns and adherence to current antibiotic guidelines. METHODS: Medical records from 2010 to 2018 for cIAIs were examined. Complicated appendicitis and complicated diverticulitis cases were included. Exclusion criteria included other etiologies of IAIs, pediatric cases, and cancer operations. RESULTS: Fifty-nine complicated appendicitis cases and 96 complicated diverticulitis cases were identified. For all cases, antibiotic duration prior to publication of the SIS guidelines was significantly longer than post-SIS duration (appendicitis: 12.6 ± 1.1 days pre-SIS [n = 37] vs 9.0 ± 1.1 days post-SIS [n = 22], P = .01; diverticulitis: 15.1 ± 0.8 days pre-SIS [n = 49] vs 11.2 ± 0.5 post-SIS [n = 47], P = .04). Surgical management (SM) was associated with shorter duration of postsource control antibiotic exposure compared with percutaneous drainage (PD) for both appendicitis (SM 10.0 ± 1.2 days vs PD 13.4 ± 1.0 days, P = .02) and diverticulitis (SM 12.8 ± 1.5 days vs PD 16.0 ± 1.5, P = .07). Patients with complicated appendicitis received shorter duration of antibiotics when managed by acute care surgeons compared to general surgeons (8.4 ± 1.1 vs 11.9 ± 0.8, P = .02). CONCLUSION: Despite improvements after the SIS guidelines' publication, the antibiotic duration is still longer than recommended. Surgical intervention and management by acute care specialists were associated with a shorter duration of antibiotic exposure.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Appendicitis/complications , Diverticulitis/complications , Guideline Adherence , Intraabdominal Infections/drug therapy , Practice Patterns, Physicians' , Appendicitis/therapy , Diverticulitis/therapy , Drug Administration Schedule , Female , Humans , Intraabdominal Infections/diagnosis , Intraabdominal Infections/etiology , Male , Middle Aged , Practice Guidelines as Topic , Retrospective Studies
6.
Mol Cell ; 78(2): 261-274.e5, 2020 04 16.
Article in English | MEDLINE | ID: mdl-32155413

ABSTRACT

RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5' cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.


Subject(s)
Positive Transcriptional Elongation Factor B/genetics , RNA Polymerase II/genetics , Transcription Factors/genetics , Transcription, Genetic , Animals , Heat-Shock Response/genetics , Humans , Mice , Nucleosomes/genetics , Promoter Regions, Genetic
7.
Cell ; 175(3): 766-779.e17, 2018 10 18.
Article in English | MEDLINE | ID: mdl-30340042

ABSTRACT

The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.


Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms, Experimental/drug therapy , Positive Transcriptional Elongation Factor B/metabolism , Repressor Proteins/metabolism , Transcription Elongation, Genetic/drug effects , Transcriptional Elongation Factors/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Drosophila , Female , HCT116 Cells , HEK293 Cells , Heat-Shock Response , Humans , Male , Mice , Mice, Inbred BALB C , Protein Binding/drug effects , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA Polymerase II/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology
8.
Structure ; 26(12): 1594-1603.e4, 2018 12 04.
Article in English | MEDLINE | ID: mdl-30270175

ABSTRACT

Dpy-30 is a regulatory subunit controlling the histone methyltransferase activity of the KMT2 enzymes in vivo. Paradoxically, in vitro methyltransferase assays revealed that Dpy-30 only modestly participates in the positive heterotypic allosteric regulation of these methyltransferases. Detailed genome-wide, molecular and structural studies reveal that an extensive network of interactions taking place at the interface between Dpy-30 and Ash2L are critical for the correct placement, genome-wide, of H3K4me2 and H3K4me3 but marginally contribute to the methyltransferase activity of KMT2 enzymes in vitro. Moreover, we show that H3K4me2 peaks persisting following the loss of Dpy-30 are found in regions of highly transcribed genes, highlighting an interplay between Complex of Proteins Associated with SET1 (COMPASS) kinetics and the cycling of RNA polymerase to control H3K4 methylation. Overall, our data suggest that Dpy-30 couples its modest positive heterotypic allosteric regulation of KMT2 methyltransferase activity with its ability to help the positioning of SET1/COMPASS to control epigenetic signaling.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Histones/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Allosteric Regulation , Animals , Binding Sites , Epigenesis, Genetic , HEK293 Cells , Histone-Lysine N-Methyltransferase/metabolism , Humans , Methylation , Models, Molecular , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Yeasts/genetics , Yeasts/metabolism
9.
EMBO J ; 37(16)2018 08 15.
Article in English | MEDLINE | ID: mdl-30006452

ABSTRACT

Even though transcription factors (TFs) are central players of gene regulation and have been extensively studied, their regulatory trans-activation domains (tADs) often remain unknown and a systematic functional characterization of tADs is lacking. Here, we present a novel high-throughput approach tAD-seq to functionally test thousands of candidate tADs from different TFs in parallel. The tADs we identify by pooled screening validate in individual luciferase assays, whereas neutral regions do not. Interestingly, the tADs are found at arbitrary positions within the TF sequences and can contain amino acid (e.g., glutamine) repeat regions or overlap structured domains, including helix-loop-helix domains that are typically annotated as DNA-binding. We also identified tADs in the non-native reading frames, confirming that random sequences can function as tADs, albeit weakly. The identification of tADs as short protein sequences sufficient for transcription activation will enable the systematic study of TF function, which-particularly for TFs of different transcription activating functionalities-is still poorly understood.


Subject(s)
Drosophila Proteins , Trans-Activators , Transcription, Genetic , Animals , Cell Line , Drosophila Proteins/biosynthesis , Drosophila Proteins/genetics , Drosophila melanogaster , Protein Domains , Trans-Activators/biosynthesis , Trans-Activators/genetics
10.
J Surg Educ ; 75(5): 1403-1409, 2018.
Article in English | MEDLINE | ID: mdl-29650483

ABSTRACT

OBJECTIVE: In spite of the recognized benefits of ultrasound, many physicians have little experience with using ultrasound to perform procedures. Many medical schools and residency programs lack a formal ultrasound training curriculum. We describe an affordable ultrasound training curriculum and versatile, inexpensive practice model. DESIGN: Participants underwent a didactic session to teach the theory required to perform ultrasound-guided procedures. Motor skills were taught using a practice model incorporating analogs of common anatomic and pathologic structures into an opacified gelatin substrate. SETTING: The Marcia and Eugene Applebaum Simulation Learning Institute, Beaumont Hospital, Royal Oak, MI; a private nonprofit tertiary care hospital associated with the OUWB School of Medicine, Rochester, MI. PARTICIPANTS: The model was tested in a cohort of 50 medical students and general surgery residents. RESULTS: The gelatin model can be constructed for $1.03 per learner. The solid, cystic, and vascular structural analogs were readily identifiable on ultrasound and easily differentiated based on their echotextures. Eighty-four percent of participants successfully aspirated the cystic structure, 88% successfully biopsied a portion of the solid structure, and 76% successfully cannulated the tubular structure. Overall, 82% of participants achieved a passing score for the exercise based on a validated Objective Structured Assessment of Technical Skill instrument. There were no significant differences between the medical students and residents. CONCLUSION: This model can be used to teach basic ultrasound skills such as aspiration, biopsy, and vessel cannulation, providing a foundation for the use of ultrasound in a broad range of clinical procedures, as well as providing practice opportunities for medical students and residents to gain increased ultrasound competency and confidence.


Subject(s)
Education, Medical, Graduate/methods , Education, Medical, Undergraduate/methods , Image-Guided Biopsy , Surgery, Computer-Assisted , Ultrasonography, Interventional , Cost-Benefit Analysis , Curriculum , Educational Measurement , Female , Gelatin , Humans , Internship and Residency/methods , Male , Models, Anatomic , Models, Educational , Students, Medical/statistics & numerical data , Ultrasonography , United States
11.
J Surg Educ ; 75(5): 1389-1394, 2018.
Article in English | MEDLINE | ID: mdl-29433996

ABSTRACT

OBJECTIVE: Our aim was to develop an ultrasound-guided training curriculum for continuous infusion catheter placement in the paravertebral space and to create a gelatin thoracic spine-rib model for use in this training. We sought to create a model that was inexpensive and reusable such that multiple participants could use one model during training. DESIGN: The model was prepared by embedding a firm foam thoracic spine replica with bilateral attached ribs into an opaque gelatin mixture. Once solidified, a preselected area was excised on each side, such that the model could be easily refilled with new gelatin blocks for use by each participant. This allowed for multiple participants to use the same model while eliminating confusion with prior tract marks. SETTING: The Marcia and Eugene Applebaum Simulation Learning Institute, Beaumont Hospital, Royal Oak, MI; a private nonprofit tertiary care hospital associated with the OUWB School of Medicine, Rochester, MI. PARTICIPANTS: Fifty-two medical students and general surgery residents underwent a 30-minute didactic session on ultrasound technique for catheter placement followed by practice on the gelatin model. RESULTS: The texture and echogenicity of the model were subjectively comparable to those of tissue in vivo and the osseous elements of the spine in the model were clearly identified using ultrasound. The exchangeable catheter placement area provided an efficient and effective method to test accurate performance in catheter placement by multiple users. Participants increased their confidence in the use of ultrasound for this procedure. CONCLUSIONS: To date, this is the first gelatin thoracic spine-rib model that has been used to teach ultrasound-guided catheter insertion into the paravertebral space, with removable testing areas that can be used by multiple users. This model can provide an inexpensive training tool that can be used in a surgical simulation setting.


Subject(s)
Catheterization/methods , Clinical Competence , Education, Medical, Graduate/methods , Models, Anatomic , Spine , Ultrasonography, Interventional , Catheters, Indwelling , Female , Gelatin , Humans , Internship and Residency/methods , Male , Models, Educational , Simulation Training/methods , Students, Medical/statistics & numerical data , Surgery, Computer-Assisted/education
12.
Nat Methods ; 15(2): 141-149, 2018 02.
Article in English | MEDLINE | ID: mdl-29256496

ABSTRACT

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.


Subject(s)
Chromatin/genetics , Enhancer Elements, Genetic , Gene Expression Regulation , Genes, Reporter , Promoter Regions, Genetic , Chromosome Mapping , False Negative Reactions , Genome, Human , HeLa Cells , Humans
13.
Nat Med ; 23(4): 493-500, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28263307

ABSTRACT

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise. A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M). Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.


Subject(s)
Brain Stem Neoplasms/genetics , Chromatin/metabolism , Gene Expression Regulation, Neoplastic/genetics , Glioma/genetics , Histone Code/genetics , Histones/genetics , Nucleosomes/metabolism , Polycomb Repressive Complex 2/metabolism , RNA-Binding Proteins/metabolism , Acetylation/drug effects , Animals , Azepines/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Chromatin/drug effects , Epigenomics , Gene Expression Regulation, Neoplastic/drug effects , Histone Code/drug effects , Histones/drug effects , Humans , Methylation/drug effects , Mice , Molecular Targeted Therapy , Mutation , Neurogenesis/drug effects , Neurogenesis/genetics , Nucleosomes/drug effects , Polycomb Repressive Complex 2/drug effects , Protein Transport , RNA-Binding Proteins/antagonists & inhibitors , Triazoles/pharmacology , Xenograft Model Antitumor Assays
14.
Cell ; 168(1-2): 59-72.e13, 2017 Jan 12.
Article in English | MEDLINE | ID: mdl-28065413

ABSTRACT

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.


Subject(s)
Leukemia, Biphenotypic, Acute/drug therapy , Leukemia, Biphenotypic, Acute/metabolism , Proteolysis/drug effects , Animals , Disease Models, Animal , Histone-Lysine N-Methyltransferase/metabolism , Humans , Interleukin-1/metabolism , Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Interleukin-1 Receptor-Associated Kinases/metabolism , Mice , Mice, Inbred C57BL , Myeloid-Lymphoid Leukemia Protein/metabolism , Ubiquitin-Conjugating Enzymes
15.
Mol Cell ; 63(2): 318-328, 2016 07 21.
Article in English | MEDLINE | ID: mdl-27447986

ABSTRACT

Polycomb response elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs in mammals remains debated. Accumulating evidence supports a model in which CpG islands recruit Polycomb group (PcG) complexes; however, which subset of CGIs is selected to serve as PREs is unclear. Trithorax (Trx) positively regulates gene expression in Drosophila and co-occupies PREs to antagonize Polycomb-dependent silencing. Here we demonstrate that Trx-dependent H3K4 dimethylation (H3K4me2) marks Drosophila PREs and maintains the developmental expression pattern of nearby genes. Similarly, the mammalian Trx homolog, MLL1, deposits H3K4me2 at CpG-dense regions that could serve as PREs. In the absence of MLL1 and H3K4me2, H3K27me3 levels, a mark of Polycomb repressive complex 2 (PRC2), increase at these loci. By inhibiting PRC2-dependent H3K27me3 in the absence of MLL1, we can rescue expression of these loci, demonstrating a functional balance between MLL1 and PRC2 activities at these sites. Thus, our study provides rules for identifying cell-type-specific functional mammalian PREs within the human genome.


Subject(s)
Chromosomal Proteins, Non-Histone/genetics , Colorectal Neoplasms/genetics , CpG Islands , DNA Methylation , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Evolution, Molecular , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Response Elements , Animals , Chromosomal Proteins, Non-Histone/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Neoplastic , HCT116 Cells , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Histones/metabolism , Humans , Myeloid-Lymphoid Leukemia Protein/metabolism , RNA Interference , Species Specificity , Transcription, Genetic , Transfection
16.
Genes Dev ; 30(1): 92-101, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26728555

ABSTRACT

Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer.


Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation , Genomic Imprinting/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Iodide Peroxidase/genetics , Iodide Peroxidase/metabolism , Alleles , Animals , Calcium-Binding Proteins , Cell Line , Chromatin Immunoprecipitation , DNA Methylation , Embryonic Stem Cells , Gene Silencing , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Sequence Analysis, DNA
17.
Mol Cell ; 60(3): 435-45, 2015 Nov 05.
Article in English | MEDLINE | ID: mdl-26527278

ABSTRACT

Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.


Subject(s)
DNA Polymerase II/metabolism , Mitosis/physiology , Positive Transcriptional Elongation Factor B/metabolism , Transcription Elongation, Genetic/physiology , Transcriptional Activation/physiology , HEK293 Cells , HeLa Cells , Humans
18.
Cell ; 162(5): 1003-15, 2015 Aug 27.
Article in English | MEDLINE | ID: mdl-26279188

ABSTRACT

The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify that Pol-II-associated factor 1 (PAF1) possesses an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II's C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase super elongation complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing PAF1 as a regulator of promoter-proximal pausing by Pol II.


Subject(s)
Nuclear Proteins/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Transcription, Genetic , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster , Histones/metabolism , Humans , Phosphorylation , RNA Interference , Transcription Factors , Ubiquitination
19.
Genes Dev ; 28(2): 115-20, 2014 Jan 15.
Article in English | MEDLINE | ID: mdl-24402317

ABSTRACT

The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner.


Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Saccharomyces cerevisiae/enzymology , Lysine/metabolism , Membrane Proteins/metabolism , Methylation , Phosphoric Monoester Hydrolases/metabolism , Protein Stability , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism
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