Biology subjects

Fields, P. E.

Publications and source records attributed to Fields, P. E..

7 recordsLinked to original sources

bioRxiv2025

mRNA isoform switching plays a crucial role in mural cumulus differentiation

During gonadotropin-induced ovarian follicle development, an antral cavity forms within the layers of granulosa cells (GCs). Gonadotropin stimulation also differentiates the GCs into two distinct lineages: mural GCs (mGCs), which surround the antral cavity, and cumulus GCs (cGCs), which stay in contact with the oocyte. We examined the transcriptomes of mouse mural and cumulus cells to understand the mechanism of differentiation. In addition to analyzing a single transcript expression per gene, we also considered multiple isoform expressions to explore differential transcriptomics. GC-specific core transcription factors Foxl2, Nr5a1, Nr5a2, Runx1, and Runx2 were expressed at high levels in mGCs but downregulated in cGCs, indicating that cGCs acquire a more differentiated state. Both single-transcript and multiple-isoform analyses revealed differential expression of about 70% of transcripts between mGCs and cGCs. Although the counts were similar, the differentially expressed genes (DEGs) at the single transcript level did not correlate well with the respective differentially expressed transcript isoforms (DETI). We identified DETIs originating from key epigenetic and transcriptional regulator genes, such as Chd1, Ezh2, Kdm5a, Kdm5b, Gata4, Esr2, Fos, Myc, and Ybx1, that were not differentially expressed at the single-transcript analysis. Further analysis revealed a transcript switch in one-third of the DETIs. Most of the transcript isoforms were protein-coding, followed by non-coding regulatory RNAs. A total of 1,302 transcript isoforms were silenced in cGCs, including those of Adar, Cebpa, Dnmt3a, Foxo4, Pgr, Rest, Runx1, Satb2, Sirt1, Sirt2, and Tead1. Conversely, 529 transcript isoforms were activated in cGCs, including transcripts for Brd7, Crem, Chd1, Med21, Med27, Nfkbia, Rbm39, Rbmx, Suv39h2, Tcf12, Xist, and Ybx3. Additionally, 57 genes exhibited DETIs, with at least one isoform turned off and another turned on in cGCs, including Csde1, Dab2, Ezh2, Gata4, Gnas, Gtf2i, Macf1, Klf10, Setdb1, and Sp3. Finally, we explored the mechanisms underlying transcript switching during the differentiation of mGCs and cGCs. Our findings suggest that gonadotropin-induced transcript switching in GCs is crucial for mural and cumulus granulosa differentiation, a key insight that would be missed without mRNA isoform analysis.

cell biology↗

bioRxiv2025

Aging alters mRNA processing in the mouse ovary

Aging in females predominantly impacts the ovaries before any other organ systems. This has profound implications for womens reproductive health. This phenomenon is closely linked to a gradual depletion of the ovarian follicle reserve and a notable diminishment of oocyte quality. Studies have shown that cellular changes within ovaries can manifest even before the observable depletion of ovarian follicles. To understand the molecular mechanisms underlying these changes, we have conducted a comprehensive analysis of the changes in gene expression in aging mouse ovaries. Using efficient genomics software such as CLC Genomic Workbench, we could detect not only the differentially expressed genes but also delineate the various transcript variants present in the transcriptome of aging ovaries. We verified the results by comparing coding sequences of selected transcripts with the coding sequences of their canonical counterparts from young and aged mice. In general, the analysis methods yielded similar observations. Our findings revealed that traditional gene expression analyses often overlook the differential expression of numerous transcript variants. We identified significant alterations in the expression patterns of alternative transcripts in aging ovaries and found coding sequences that lead to profound functional outcomes. Notably, most of these differentially expressed transcript variants were affected upstream epigenetically and transcriptionally, then generated through alternative splicing events. This suggests that aging may lead to alterations in RNA-binding proteins and spliceosome components, which play a crucial role in mRNA processing within the mouse ovary. Our observations highlight the necessity of focusing on transcript variants and their functions in aging research, as they provide a more nuanced understanding of the biological processes at play.

molecular biology↗

bioRxiv2024

Epigenetic and transcriptional regulation of ovarian development altered in ErβKO ovaries

We analyzed the transcriptome of wildtype and estrogen receptor {beta} knockout (Er{beta}KO) rat ovaries during the early postnatal period and detected remarkable changes in epigenetic regulators and transcription factors. Compared to postnatal day (PD) 4.5 wildtype ovaries, 17 differentially expressed epigenetic regulators (DEERs), and 23 differentially expressed transcription factors (DETFs) were detected in PD6.5 wildtype ovaries. Subsequently, compared to PD 6.5 wildtype ovaries, 24 DEERs and 68 DETFs were detected in PD8.5 ovaries. Changes in DEERs and DEFTs resulted in 581 differentially expressed downstream genes (DEDGs) in PD6.5 and 920 DEDGs in wildtype PD8.5 ovaries. The DEERs, DETFs, and DEDGs in wildtype ovaries represented primordial follicle activation (PFA) and development of the first-wave follicles because the second-wave follicles remain dormant during this period. However, the changes in DEERs, DETFs, and DEDGs during this postnatal period were markedly different in Er{beta}KO rat ovaries, which suffered from increased PFA in both waves. Compared to 17 DEERs and 23 DETFs in wildtype, 46 DEERs and 55 DETFs were identified in PD 6.5 Er{beta}KO ovaries. The differences were more remarkable in PD 8.5 Er{beta}KO ovaries; compared to 24 DEERS and 68 DETFs in wildtype, only 8 DEERs and 10 DETFs were detected in Er{beta}KO ovaries. Such dysregulation resulted in altered DEDGs in PD 6.5 (581 vs. 744) and in PD8.5 (920 vs. 191) Er{beta}KO ovaries. These findings also suggest that the number of DEDGs depends directly on the numbers of DEERS and DETFs. In addition to the quantitative differences in DEERs and DETFs between the wildtype and Er{beta}KO ovaries, we detected distinct differences in the identities of the regulators. Our observations indicate that loss of ER{beta} dysregulates the epigenetic regulators and transcription factors in Er{beta}KO ovaries, which disrupts the downstream genes in ovarian follicles and increases follicle activation.

developmental biology↗

bioRxiv2024

Importance of transcript variants in transcriptome analyses

RNA sequencing (RNA-Seq) has become a widely adopted genome-wide technique for investigating gene expression patterns. However, conventional RNA-Seq analyses typically rely on gene expression (GE) values that aggregate all the transcripts produced by a gene under a single identifier, overlooking the complexity of transcript variants arising from different transcription start sites and alternative splicing events. In this study, we explored the implications of neglecting transcript variants in RNA-Seq analyses. Among the 1334 transcription factor (TF) genes expressed in mouse embryonic stem (ES) or trophoblast stem (TS) cells, 652 were reported to be differentially expressed in TS cells based on GE values (365 upregulated and 287 downregulated, [≥]2-fold, FDR p-value [≤]0.05). Intriguingly, differential gene expression analysis revealed that of the 365 upregulated genes, 883 transcript variants were expressed, with only 174 (<20%) variants exhibiting upregulation based on transcript expression (TE) values. The remaining 709 (>80%) variants were either down-regulated or showed no significant change in expression analysis. Similarly, the 287 genes reported to be downregulated expressed 856 transcript variants, with only 153 (<20%) downregulated variants and 703 (>82%) variants that were upregulated or showed no significant changes. Additionally, the 682 TF genes that did not show significant changes between ES and TS cells (GE values < 2-fold changes and/or FDR p-values >0.05) expressed 2215 transcript variants, which included 477 (>21%) that were differentially expressed (276 upregulated and 201 downregulated, [≥]2-fold, FDR p-value [≤]0.05). Notably, a particular gene does not express just one protein; rather its transcript variants encode multiple proteins with distinct functional domains, including non-coding regulatory RNAs. Our findings underscore the critical necessity of considering transcript variants in RNA-Seq analyses. Doing so may enable a more precise understanding of the intricate functional and regulatory landscape of genes; ignoring the variants may result in an erroneous interpretation. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/603122v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1371b9corg.highwire.dtl.DTLVardef@f5c037org.highwire.dtl.DTLVardef@1e5f79org.highwire.dtl.DTLVardef@e20cb_HPS_FORMAT_FIGEXP M_FIG C_FIG Differential expression of transcription factors (TFs) between mouse embryonic stem (ES) cells and trophoblast stem (TS) cells. This graphic presentation clearly demonstrates the importance of including transcript variants during RNA sequencing (RNA-Seq) analyses. Panel A represents the conventional differential gene expression analysis approach after RNA-Seq, where all transcript reads are taken under a single gene name. Panel B takes differential gene expression analysis one step further by examining all the transcript variants that were previously hidden under the main gene name. Our results indicate that exclusive gene expression (GE) analysis inaccurately defines over 80% of the transcript expression (TE). Without analyses of all the transcript variants reads, we fail to uncover the functional importance of the variants and the regulation of their expression. Both GE and TE values are expressed as transcript per million (TPM). Data analyses were performed by using CLC Genomics Workbench.

bioinformatics↗

bioRxiv2020

DOT1L primarily acts as a transcriptional repressor in hematopoietic progenitor cells

DOT1L is essential for early hematopoiesis but the precise mechanisms remain largely unclear. The only known function of DOT1L is histone H3 lysine 79 (H3K79) methylation. We generated two mouse models; a Dot1L-knockout (Dot1L-KO), and another possessing a point mutation in its methyltransferase domain (Dot1L-MM) to determine the role of its catalytic activity during early hematopoiesis. We observed that Dot1L-KO embryos suffered from severe anemia, while Dot1L-MM embryos showed minimal to no anemia. However, ex vivo culture of Dot1L-MM hematopoietic progenitors (HPCs) exhibited defective development of myeloid and mixed progenitors. DOT1L is a well-recognized, cell-type specific epigenetic regulator of gene expression. To elucidate the mechanisms underlying such diverse hematopoietic properties of Dot1L-KO and Dot1L-MM HPCs, we examined their whole transcriptomes. Extensively self-renewing erythroblast (ESRE) cultures were established using yolk sac (YS) cells collected on embryonic day 10.5 (E10.5). Dot1l-KO and Dot1l-MM cells expanded significantly less than the wildtype cells and showed slower progression through the cell cycle. Total RNA extracted from the wildtype and Dot1l-mutant ESRE cells were subjected to RNA-seq analyses. We observed that the majority (~82%) of the differentially expressed genes (DEGs) were upregulated in both of the Dot1L-mutants, which suggests that DOT1L predominantly acts as a transcriptional repressor in HPCs. We also observed that about ~40% of the DEGs were unique to either of the mutant group, suggesting that DOT1L possesses both methyltransferase domain-dependent and -independent functions. We further analyzed Gene Ontology and signaling pathways relevant to the DEGs common to both mutant groups and those that were unique to either group. Among the common DEGs, we observed upregulation of CDK inhibitors, which explains the cell cycle arrest in both of the Dot1L-mutant progenitors.

developmental biology↗

bioRxiv2020

DOT1L methyltransferase regulates the calcium influx in erythroid progenitor cells in response to erythropoietin

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of hematopoietic progenitor cells. An important downstream response of EPO signaling is calcium influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in c-KIT positive erythroid progenitor cells is regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development, including early erythropoiesis. We previously reported that Dot1L knockout (Dot1L-KO) hematopoietic progenitors in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we have detected a marked downregulation of Trpc6 gene expression in Dot1L-KO progenitor cells in the yolk sac compared to wildtype. However, the expression of Trpc2, the positive regulator of Ca2+influx, remained unchanged. The promoter and the proximal region of the Trpc6 gene loci exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. As the loss of DOT1L affects the expression of TRPC6, which inhibits Ca2+influx by TRPC2, Dot1L-KO progenitor cells in the yolk sac exhibit accelerated and sustained high levels of Ca2+influx. Such heightened Ca2+ levels might have detrimental effects on the development of hematopoietic progenitor cells in response to erythropoietin.

developmental biology↗

bioRxiv2020

The Role of DOT1L Methyltransferase Activity in Fetal Hematopoiesis

Early mammalian erythropoiesis requires the DOT1L methyltransferase. We demonstrated that loss of DOT1L in mutant mice resulted in lethal anemia during midgestation. The molecular mechanisms by which DOT1L regulates embryonic erythropoiesis have not yet been elucidated. In this study, a methyltransferase mutant mouse line (Dot1L-MM) was generated to determine whether the methyltransferase activity of DOT1L is essential for erythropoiesis. Dot1L-MM mice displayed embryonic lethality between embryonic days 10.5 and 13.5, similar to Dot1lL knockout (Dot1L-KO) mice. However, when examined at E10.5, unlike the Dot1L-KO, Dot1L-MM embryos did not exhibit evidence of anemia. In ex vivo hematopoietic differentiation cultures, Dot1L-KO and Dot1L-MM yolk sac (YS) cells both formed reduced numbers of myeloid, and mixed hematopoietic colonies. Erythroid colonies were able to be formed in numbers equal to wildtype embryos. Extensively self-renewing erythroblast (ESRE) cultures were established using YS cells from E10.5 embryos. Dot1L-KO and Dot1L-MM cells expanded significantly less than wild-type cells and exhibited increased cell death. Strikingly, Dot1L-KO and Dot1L-MM cells of YS origin exhibited profound genomic instability, implicating DOT1L methyltransferase activity in maintenance of the genome as well as viability of hematopoietic progenitors. Our results indicate that the methyltransferase activity of DOT1L plays an important role early murine hematopoiesis.

developmental biology↗