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The current academic culture facing women in science, technology, engineering, and math (STEM) fields in the United States has sparked the formation of grassroots advocacy groups to empower female scientists in training. However, the impact of these initiatives often goes unmeasured and underappreciated. Our Women in Science and Engineering (WiSE) organization serves postdoctoral researchers, graduate students, and research technicians (trainees) at a private research institute for biological sciences. Here we propose the following guidelines for cultivating a successful women-in-STEM-focused group based upon survey results from our own scientific community as well as the experience of our WiSE group leaders. We hope these recommendations can provide guidance to advocacy groups at other research and academic organizations that wish to strengthen their efforts. Whereas our own group specifically focuses on the underrepresented state of women in science, we hope these guidelines may be adapted and applied to groups that advocate for any minority group within the greater scientific community (i.e., those of gender, race/ethnicity, socioeconomic background, sexual orientation, etc.)., Author summary Women are underrepresented in leadership positions in the fields of science, technology, engineering, and math (STEM) despite obtaining undergraduate and graduate-level degrees at roughly the same rate as men. To take advantage of this resource of highly trained women in STEM fields, there needs to be better career support for female scientists in training. The authors report on their experience running a women-in-STEM-focused group to foster a more supportive, collaborative, and egalitarian scientific community. In particular, the authors use data-driven metrics to evaluate the success of their group in meeting the needs of their community. The results of this evaluation are presented as a set of 10 guidelines for establishing and maintaining advocacy groups that support the careers of women in science.

Transposable Elements (TEs) are mobile genetic elements whose sequences constitute nearly half of the human genome. Each TE copy can be present in hundreds to thousands of locations within the genome, complicating the genetic and genomic studies of these highly repetitive sequences. The recent development of better tools for evaluating TE derived sequences in genomic studies has enabled an increasing appreciation for the contribution of TEs to human development and disease. While some TEs have contributed novel and beneficial host functions, this review will summarize the evidence for detrimental TE activity in neurodegenerative disorders. Much of the evidence for pathogenicity implicates endogenous retroviruses (ERVs), a subset of TEs that entered the genome by retroviral infections of germline cells in our evolutionary ancestors and have since been passed down as a substantial fraction of the human genome. Human specific ERVs (HERVs) represent some of the youngest ERVs in the genome, and thus are presumed to retain greater function and resultant pathogenic potential.

SummaryAmyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several inherited pathogenic mutations have been identified as causative, the vast majority of cases are sporadic with no family history of disease. Thus, for the majority of ALS cases, a specific causal abnormality is not known and the disease may be a product of multiple inter-related pathways contributing to varying degrees in different ALS patients. Using unsupervised machine learning algorithms, we stratified the transcriptomes of 148 ALS decedent cortex tissue samples into three distinct and robust molecular subtypes. The largest cluster, identified in 61% of patient samples, displayed hallmarks of oxidative and proteotoxic stress. Another 20% of the ALS patient samples exhibited high levels of retrotransposon expression and other signatures of TDP-43 dysfunction. Finally, a third group showed predominant signatures of glial activation (19%). Together these results demonstrate that at least three distinct molecular signatures contribute to ALS disease. While multiple dysregulated components and pathways comprising these clusters have previously been implicated in ALS pathogenesis, unbiased analysis of this large survey demonstrated that sporadic ALS patient tissues can be segregated into distinct molecular subsets.

The single cell is considered the basic unit of biology, and the pursuit of understanding how heterogeneous populations of cells can functionally coexist in tissues, organisms, microbial ecosystems, and even cancer, makes them the subject of intense study. Next-generation sequencing (NGS) of RNA and DNA has opened a new frontier of (single)-cell biology. Hundreds to millions of cells now can be assayed in parallel, providing the molecular profile of each cell in its milieu inexpensively and in a manner that can be analyzed mathematically. The goal of this article is to provide a high-level overview of single-cell sequencing for the nonexpert and show how its applications are influencing both basic and applied clinical studies in embryology, developmental genetics, and cancer.

Next-generation sequencing approaches have fundamentally changed the types of questions that can be asked about gene function and regulation. With the goal of approaching truly genome-wide quantifications of all the interaction partners and downstream effects of particular genes, these quantitative assays have allowed for an unprecedented level of detail in exploring biological interactions. However, many challenges remain in our ability to accurately describe and quantify the interactions that take place in those hard to reach and extremely repetitive regions of our genome comprised mostly of transposable elements (TEs). Tools dedicated to TE-derived sequences have lagged behind, making the inclusion of these sequences in genome-wide analyses difficult. Recent improvements, both computational and experimental, allow for the better inclusion of TE sequences in genomic assays and a renewed appreciation for the importance of TE biology. This review will discuss the recent improvements that have been made in the computational analysis of TE-derived sequences as well as the areas where such analysis still proves difficult. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

MicroRNAs (miRNAs) are small 21-22nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various small RNA classes remains unclear, largely because these small RNAs are typically analyzed separately. Here we present TEsmall, a tool that allows for the simultaneous processing and analysis of small RNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to small RNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other small RNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.

Chd5 loss links the up-regulation of ribosomal genes to enhanced translation, causing the untimely production of a master transcription factor that unleashes stem cells and alters cell fate., Neural cell fate specification is a multistep process in which stem cells undergo sequential changes in states, giving rise to particular lineages such as neurons and astrocytes. This process is accompanied by dynamic changes of chromatin and in transcription, thereby orchestrating lineage-specific gene expression programs. A pressing question is how these events are interconnected to sculpt cell fate. We show that altered chromatin due to loss of the chromatin remodeler Chd5 causes neural stem cell activation to occur ahead of time. This premature activation is accompanied by transcriptional derepression of ribosomal subunits, enhanced ribosome biogenesis, and increased translation. These untimely events deregulate cell fate decisions, culminating in the generation of excessive numbers of astrocytes at the expense of neurons. By monitoring the proneural factor Mash1, we further show that translational control is crucial for appropriate execution of cell fate specification, thereby providing new insight into the interplay between transcription and translation at the initial stages of neurogenesis.

SummaryPancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

SummaryChromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD., Author summary Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, and mutations in TDP-43 are causal in a subset of familial ALS cases. Although cytoplasmic inclusions of this mostly nuclear protein are a hallmark of the disease, the cascade of events leading to cell death are not understood. We demonstrate that expression of human TDP-43 (hTDP-43) in Drosophila neurons or glial cells, which results in toxic cytoplasmic accumulation of TDP-43, causes broad expression of retrotransposons. In the case of glial hTDP-43 expression, the endogenous retrovirus (ERV) gypsy causally contributes to degeneration because inhibiting gypsy genetically or pharmacologically is sufficient to rescue the phenotypic effects. Moreover, we demonstrate that activation of DNA damage-mediated programmed cell death underlies hTDP-43 and gypsy mediated toxicity. Finally, we find that hTDP-43 pathology impairs small interfering RNA silencing, which is an essential system that normally protects the genome from RTEs. These findings suggest a novel mechanism in which a storm of retrotransposon activation drives neurodegeneration in TDP-43 mediated diseases such as ALS and FTLD.

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer., The p53-inducible antiproliferative gene BTG2 is suppressed in many cancers, in the absence of inactivating gene mutations. Here the authors show that the histone lysine methyltransferase SETD1A suppresses the expression of several p53 target genes including BTG2 by inducing a network of microRNAs.

microRNAs comprise a few percent of animal genes and have been recognized as important regulators of a diverse range of biological processes. Understanding the biological functions of miRNAs requires effective means to identify their targets. Combined efforts from computational prediction, miRNA over-expression or depletion, and biochemical purification have identified thousands of potential miRNA-target pairs in cells and organisms. Complementarity to the miRNA seed sequence appears to be a common principle in target recognition. Other features, including miRNA-target duplex stability, binding site accessibility, and local UTR structure might affect target recognition. Yet computational approaches using such contextual features have yielded largely nonoverlapping results and experimental assessment of their impact has been limited. Here, we compare two large sets of miRNA targets: targets identified using an improved Ago1 immunopurification method and targets identified among transcripts up-regulated after Ago1 depletion. We found surprisingly limited overlap between these sets. The two sets showed enrichment for target sites with different molecular, structural and functional properties. Intriguingly, we found a strong correlation between UTR length and other contextual features that distinguish the two groups. This finding was extended to all predicted microRNA targets. Distinct repression mechanisms could have evolved to regulate targets with different contextual features. This study reveals a complex relationship among different features in miRNA-target recognition and poses a new challenge for computational prediction.

Elevated expression of specific transposable elements (TEs) has been observed in several neurodegenerative disorders. TEs also can be active during normal neurogenesis. By mining a series of deep sequencing datasets of protein-RNA interactions and of gene expression profiles, we uncovered extensive binding of TE transcripts to TDP-43, an RNA-binding protein central to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Second, we find that association between TDP-43 and many of its TE targets is reduced in FTLD patients. Third, we discovered that a large fraction of the TEs to which TDP-43 binds become de-repressed in mouse TDP-43 disease models. We propose the hypothesis that TE mis-regulation contributes to TDP-43 related neurodegenerative diseases.