Histones and their posttranslational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well ...established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have nonhistone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike inferences drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity but not for gene activation. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.
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•Specific histone genotypes can be engineered in Drosophila using BAC transgenes•Histone gene expression is controlled by an active dosage compensation mechanism•Posttranslational modification of H4K20 is not required to complete development•H3K27 is required for Polycomb target gene repression but not for gene activation
McKay et al. describe a platform for histone gene engineering in Drosophila that allows direct genetic assessment of histone residue function in vivo. Using the system, they demonstrate that, contrary to inferences based on analysis of H4K20 methyltransferases, H4K20 is not essential for DNA replication or completion of development.
The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) ...and U7 small nuclear RNP (snRNP) are HLB components that participate in 3' processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3' end processing with transcription termination.
Whether dietary fiber protects against colorectal cancer is controversial because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer ...have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We used gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as a histone deacetylase (HDAC) inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared with normal colonic tissues.
These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy.
The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and ...cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A
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and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions.
Background
The immunohistochemical (IHC) marker PReferentially expressed Antigen in MElanoma (PRAME) has shown promise in the diagnosis of melanocytic lesions. A few studies have investigated PRAME ...IHC expression in acral melanomas, but PRAME expression in subungual melanomas is largely unknown. We evaluated the utility of PRAME IHC expression in distinguishing subungual melanomas (SUM) and non‐subungual acral melanomas (AM) from acral nevi (AN).
Methods
Twenty‐two SUM, 20 AM, and 14 AN were identified. IHC studies were performed using an anti‐PRAME antibody. The percentage of lesional cells with PRAME expression was recorded and categorized as follows: 0%, 0; 1%–25%, 1+; 26%–50%, 2+; 51%–75%, 3+; and >75%, 4+. Patient demographics and other relevant clinicopathologic parameters were recorded.
Results
Diffuse (4+) PRAME IHC expression was identified in 55% (12/22) SUM and 70% (14/20) AM, respectively. Any PRAME expression (1+ to 4+) was identified in 73% (16/22) SUMs and 95% (19/20) AM, respectively. One of 14 (7%) AN exhibited PRAME expression; interestingly, the pattern of expression was diffuse.
Conclusions
In our study, PRAME IHC expression was useful in identifying AM, including SUM. However, there are exceptions of PRAME‐negative melanomas and PRAME‐positive nevi.
Background
Improving family engagement in juvenile justice (JJ) system behavioral health services is a high priority for JJ systems, reform organizations, and family advocacy groups across the United ...States. Family-driven care (FDC) is a family engagement framework used by youth-serving systems to elevate family voice and decision-making power at all levels of the organization. Key domains of a family-driven system of care include: 1) identifying and involving families in all processes, 2) informing families with accurate, understandable, and transparent information, 3) collaborating with families to make decisions and plan treatments, 4) responding to family diversity and inclusion, 5) partnering with families to make organizational decisions and policy changes, 6) providing opportunities for family peer support, 7) providing logistical support to help families overcome barriers to participation, and 8) addressing family health and functioning. FDC enhances family participation, empowerment, and decision-making power in youth services; ultimately, improving youth and family behavioral health outcomes, enhancing family-child connectedness, and reducing youth recidivism in the JJ setting.
Methods
We evaluated staff-perceived adoption of the eight domains of FDC across detention and community services agencies in the state of Georgia. We collected mixed methods data involving surveys and in-depth qualitative interviews with JJ system administrators, staff, and practitioners between November 2021- July 2022. In total, 140 individuals from 61 unique JJ agencies participated in surveys; and 16 JJ key informants participated in qualitative interviews.
Results
FDC domains with the highest perceived adoption across agencies included identifying and involving families, informing families, collaborative decision-making and treatment planning, and family diversity and inclusion. Other domains that had mixed or lower perceived adoption included involving families in organizational feedback and policy making, family peer support, logistical support, and family health and functioning. Adoption of FDC domains differed across staff and organizational characteristics.
Conclusions
Findings from this mixed methods assessment will inform strategic planning for the scale-up of FDC strategies across JJ agencies in the state, and serve as a template for assessing strengths and weaknesses in the application of family engagement practices in systems nationally.
Distinction between Merkel cell carcinoma (MCC) and pulmonary small cell carcinoma (PSmCC) can be challenging, even with the aid of immunohistochemistry (IHC) analysis of CK20 and TTF1, as these ...tumors occasionally lack classic immunophenotypes (CK20+/TTF1- in MCC and CK20-/TTF1+ in PSmCC).
To evaluate the diagnostic utility of SOX11 and PAX5 IHC for distinguishing MCCs from PSmCCs and compare it with that of CK20 and TTF1 IHC.
SOX11, PAX5, CK20, and TTF1 expression (pattern, intensity, and proportion of tumor cells expressing protein) was assessed in 31 primary and 16 metastatic MCCs and 20 primary and 9 metastatic PSmCCs.
SOX11 expression was present in all MCCs and was predominantly strong and diffuse. Only 19% of primary and 38% of metastatic MCCs exhibited diffuse PAX5 expression; none exhibited strong immunoreactivity. Strong and diffuse SOX11 expression was seen in less than 25% of primary and metastatic PSmCCs. PAX5 expression was rare in PSmCCs and was mostly weak and focal/patchy. SOX11 expression in at least 26% of tumor cells, with at least moderate intensity, favored the diagnosis of MCC over PSmCC (P < .001). Furthermore, SOX11 expression was more likely than CK20 expression to be strong or diffuse in sentinel lymph node (SLN) metastases of MCC, indicating that SOX11 is superior to CK20 for detecting tumor deposits in SLNs in MCC.
Our findings indicate that SOX11 not only is a powerful marker for distinguishing MCCs from PSmCCs, especially when used in conjunction with CK20 and TTF1, but also has utility for screening SLNs in MCC.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Deep penetrating nevi (DPN), particularly those showing combined features, or combined deep penetrating nevi (CDPN), may show histopathological resemblance to blue nevus (BN) and melanoma. ...Preferentially Expressed Antigen in MElanoma (PRAME) is a marker that helps distinguish melanoma from benign melanocytic lesions. Lymphoid enhancer-binding factor 1 (LEF1) has been proposed to be used in conjunction with β-catenin for diagnosis of DPN. The immunohistochemical expression of PRAME and LEF1 was evaluated in 10 DPN (including 6 CDPN and 2 DPN-like proliferations with atypical features), 16 BN (including combined and cellular BN), and 2 melanomas with features of DPN or BN. PRAME was negative in most DPN (n = 10/10, n = 9/10, one case with discrepancy between readers) and all BN (n = 16/16), while the 2 melanomas included were positive (n = 2/2). All DPN were positive for LEF1 (n = 9/9) while only a subset of BN were positive (n = 6/16, P = 0.0028; n = 5/16, P = 0.001, per both readers). LEF1 seemed to be easier to interpret than β-catenin because of its nuclear pattern of expression. The expression of LEF1 in the regular nevus component of combined BN presents a potential pitfall in practice because it may lead to misinterpretation of LEF1 as positive in the BN component of the lesion. However, a subset (approximately one-third) of combined BN seemed to show true LEF1 expression. Taking into account pitfalls in interpretation, the combinatorial panel of PRAME and LEF1, in addition to conventional histopathological features, may be useful to distinguish CDPN from combined BN and other benign and malignant mimics.
Deep penetrating nevi (DPN), particularly those showing combined features, or combined deep penetrating nevi (CDPN), may show histopathological resemblance to blue nevus (BN) and melanoma. ...Preferentially Expressed Antigen in MElanoma (PRAME) is a marker that helps distinguish melanoma from benign melanocytic lesions. Lymphoid enhancer-binding factor 1 (LEF1) has been proposed to be used in conjunction with β-catenin for diagnosis of DPN. The immunohistochemical expression of PRAME and LEF1 was evaluated in 10 DPN (including 6 CDPN and 2 DPN-like proliferations with atypical features), 16 BN (including combined and cellular BN), and 2 melanomas with features of DPN or BN. PRAME was negative in most DPN (n = 10/10, n = 9/10, one case with discrepancy between readers) and all BN (n = 16/16), while the 2 melanomas included were positive (n = 2/2). All DPN were positive for LEF1 (n = 9/9) while only a subset of BN were positive (n = 6/16, P = 0.0028; n = 5/16, P = 0.001, per both readers). LEF1 seemed to be easier to interpret than β-catenin because of its nuclear pattern of expression. The expression of LEF1 in the regular nevus component of combined BN presents a potential pitfall in practice because it may lead to misinterpretation of LEF1 as positive in the BN component of the lesion. However, a subset (approximately one-third) of combined BN seemed to show true LEF1 expression. Taking into account pitfalls in interpretation, the combinatorial panel of PRAME and LEF1, in addition to conventional histopathological features, may be useful to distinguish CDPN from combined BN and other benign and malignant mimics.
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