Biology subjects

Macfarlan, T.

Publications and source records attributed to Macfarlan, T..

6 recordsLinked to original sources

bioRxiv2025

Pachytene piRNAs define a conserved program of meiotic gene regulation

PIWI-interacting RNAs (piRNAs) safeguard genome integrity and fertility across animals and in humans1-3. In mammals, pre-pachytene piRNAs execute the ancestral program of transposon silencing, whereas a second, mammalian-specific class functions during meiosis4. These pachytene piRNAs are indispensable for spermatogenesis3, yet their sequence diversity, rapid evolution, and lack of obvious complementarity have long obscured their primary targets and mode of action5. Here, we show that groups of pachytene piRNAs converge on single mRNAs with near-perfect complementarity, evoking the specificity and efficacy of classical RNA interference6. These mRNA-targeting piRNAs originate from pseudogene fragments within discrete piRNA clusters, establishing an unexpected one-to-one relationship: a given cluster encodes thousands of piRNAs that collectively silence a single gene. Deleting such piRNA clusters or their embedded pseudogene fragments, eliminates the gene-targeting piRNAs, derepresses the target mRNA, and causes spermatogenic defects. Comparative genomics reveals a simple, unifying logic, whereby rapidly evolving cluster-target pairs coexist with a conserved, primate-specific regulon maintained over tens of millions of years. These findings provide a mechanistic framework for pachytene piRNA action, repositioning them as precise gene regulators during meiosis and highlighting a uniquely preserved piRNA-target pair in primates.

developmental biology↗

bioRxiv2022

AGO2 silences mobile transposons in the nucleus of quiescent cells

Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway with essential roles in development and disease. Yet, little is known about its regulation in vivo. Here, we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells where mTOR signaling is low, AGO2 accumulates in the nucleus where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.

molecular biology↗

bioRxiv2021

Homeobox transcription factor MNX1 is crucial for restraining the expression of pan-neuronal genes in motor neurons

Motor neurons (MNs) control muscle movement and are essential for breathing, walking and fine motor skills. Motor Neuron and Pancreas Homeobox 1 (MNX1) has long been recognized as a key marker of the MN lineage. Deficiency of the Mnx1 gene in mice results in early postnatal lethality - likely by causing abnormal MN development and respiratory malfunction. However, the genome-wide targets and exact regulatory function of Mnx1 in MNs remains unresolved. Using an in vitro model for efficient MN induction from mouse embryonic stem cells, we identified about six thousand MNX1-bound loci, of which half are conserved enhancers co-bound by the core MN-inducing factors ISL1 and LHX3, while the other half are promoters for housekeeping-like genes. Despite its widespread binding, disruption of Mnx1 affects the activity of only a few dozen MNX1-bound loci, and causes mis-regulation of about one hundred genes, the majority of which are up-regulated pan-neuronal genes with relatively higher expression in the brain compared to MNs. Integration of genome-wide binding, transcriptomic and epigenomic data in the wild-type and Mnx1-disrupted MNs predicts that Pbx3 and Pou6f2 are two putative direct targets of MNX1, and both are homeobox transcription factors highly expressed in the central nervous system. Our results suggest that MNX1 is crucial for restraining the expression of many pan-neuronal genes in MNs, likely in an indirect fashion. Further, the rarity of direct targets in contrast to the widespread binding of MNX1 reflects a distinctive mode of transcriptional regulation by homeobox transcriptional factors.

molecular biology↗

bioRxiv2020

Deciphering the evolution of the transcriptional and regulatory landscape in human placenta

In mammals, the placenta mediates maternal-fetal nutrient and waste exchange and provides immunomodulatory actions that facilitate maternal-fetal tolerance. The placenta is highly diversified among mammalian species, yet the molecular mechanisms that distinguish the placenta of human from other mammals are not fully understood. Using an interspecies transcriptomic comparison of human, macaque, and mouse term placentae, we identified hundreds of genes with lineage-specific expression - including dozens that are placentally-enriched and potentially related to pregnancy. We further annotated the enhancers for different human tissues using epigenomic data and demonstrate that the placenta and chorion are unique in that their enhancers display the least conservation. We identified numerous lineage-specific human placental enhancers, and found they are highly overlapped with specific families of endogenous retroviruses (ERVs), including MER21A, MER4A/B and MER39B that were previously linked to immune response and placental function. Among these ERV families, we further demonstrate that MER41 insertions create dozens of lineage-specific Serum Response Factor (SRF) binding loci in human, including one adjacent to FBN2, a placenta-specific gene with increased expression in humans that produces the peptide hormone placensin to stimulate glucose secretion and trophoblast invasion. Our results demonstrate the prevalence of lineage-specific human placental enhancers which are frequently associated with ERV insertions and likely facilitated the lineage-specific evolution of the mammalian placenta.

evolutionary biology↗

bioRxiv2020

Non-essential function of KRAB zinc finger gene clusters in retrotransposon suppression

The Kruppel-associated box zinc finger protein (KRAB-ZFP) family amplified and diversified in mammals by segmental duplications, but the function of the majority of this gene family remains largely unexplored due to the inaccessibility of the gene clusters to conventional gene targeting. We determined the genomic binding sites of 61 murine KRAB-ZFPs and genetically deleted in mouse embryonic stem (ES) cells five large KRAB-ZFP gene clusters encoding nearly one tenth of the more than 700 mouse KRAB-ZFPs. We demonstrate that clustered KRAB-ZFPs directly bind and silence retrotransposons and block retrotransposon-borne enhancers from gene activation in ES cells. Homozygous knockout mice generated from ES cells deleted in one of two KRAB-ZFP clusters were born at sub-mendelian frequencies in some matings, but heterozygous intercrosses could also yield knockout progeny with no overt phenotype. We further developed a retrotransposon capture-sequencing approach to assess mobility of the MMETn family of endogenous retrovirus like elements, which are transcriptionally activated in KRAB-ZFP cluster KOs, in a pedigree of KRAB-ZFP cluster KO and WT mice. We identified numerous somatic and several germ-line MMETn insertions, and found a modest increase in activity in mutant animals, but these events were detected in both wild-type and KO mice in stochastic and highly variable patterns. Our data suggests that the majority of young KRAB-ZFPs play a non-essential role in transposon silencing, likely due to the large redundancy with other KRAB-ZFPs and other transposon restriction pathways in mice. One Sentence SummaryMegabase-scale deletions of KRAB-ZFP gene clusters in mice leads to retrotransposon activation.

genetics↗

bioRxiv2019

Dual Histone Methyl Reader ZCWPW1 Facilitates Repair of Meiotic Double Strand Breaks

Meiotic crossovers result from homology-directed repair of double strand breaks (DSBs). Unlike yeast and plants, where DSBs are generated near gene promoters, in many vertebrates, DSBs are enriched at hotspots determined by the DNA binding activity of the rapidly evolving zinc finger array of PRDM9 (PR domain zinc finger protein 9). PRDM9 subsequently catalyzes tri-methylation of lysine 4 and lysine 36 of Histone H3 in nearby nucleosomes. Here, we identify the dual histone methylation reader ZCWPW1, which is tightly co-expressed during spermatogenesis with Prdm9 and co-evolved with Prdm9 in vertebrates, as an essential meiotic recombination factor required for efficient synapsis and repair of PRDM9-dependent DSBs. In sum, our results indicate that the evolution of a dual histone methylation writer/reader system in vertebrates facilitated a shift in genetic recombination away from a static pattern near genes towards a flexible pattern controlled by the rapidly evolving DNA binding activity of PRDM9.

genetics↗