Paternal RLIM/Rnf12 is a survival factor for milk-producing alveolar cells.

In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), whereas extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12, which encodes the ubiquitin ligase Rnf12/RLIM. We find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy, with alveolar cells that lack RLIM undergoing apoptosis as they begin to differentiate. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to nonrandom maternal XCI in mammary epithelial cells. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and, together with population models, reveal implications of transgenerational epigenetic inheritance.

Excessive nucleic acid R-loops induce mitochondria-dependent epithelial cell necroptosis and drive spontaneous intestinal inflammation.

Oxidative stress, which can be activated by a variety of environmental risk factors, has been implicated as an important pathogenic factor for inflammatory bowel disease (IBD). However, how oxidative stress drives IBD onset remains elusive. Here, we found that oxidative stress was strongly activated in inflamed tissues from both ulcerative colitis patients and Crohn's disease patients, and it caused nuclear-to-cytosolic TDP-43 transport and a reduction in the TDP-43 protein level. To investigate the function of TDP-43 in IBD, we inducibly deleted exons 2 to 3 of Tardbp (encoding Tdp-43) in mouse intestinal epithelium, which disrupted its nuclear localization and RNA-processing function. The deletion gave rise to spontaneous intestinal inflammation by inducing epithelial cell necroptosis. Suppression of the necroptotic pathway with deletion of Mlkl or the RIP1 inhibitor Nec-1 rescued colitis phenotypes. Mechanistically, disruption of nuclear TDP-43 caused excessive R-loop accumulation, which triggered DNA damage and genome instability and thereby induced PARP1 hyperactivation, leading to subsequent NAD+ depletion and ATP loss, consequently activating mitochondrion-dependent necroptosis in intestinal epithelial cells. Importantly, restoration of cellular NAD+ levels with NAD+ or NMN supplementation, as well as suppression of ALKBH7, an α-ketoglutarate dioxygenase in mitochondria, rescued TDP-43 deficiency-induced cell death and intestinal inflammation. Furthermore, TDP-43 protein levels were significantly inversely correlated with γ-H2A.X and p-MLKL levels in clinical IBD samples, suggesting the clinical relevance of TDP-43 deficiency-induced mitochondrion-dependent necroptosis. Taken together, these findings identify a unique pathogenic mechanism that links oxidative stress to intestinal inflammation and provide a potent and valid strategy for IBD intervention.

SGCE promotes breast cancer stemness by promoting the transcription of FGF-BP1 by Sp1.

Breast cancer stem cells are mainly responsible for poor prognosis, especially in triple-negative breast cancer (TNBC). In a previous study, we demonstrated that ε-Sarcoglycan (SGCE), a type Ⅰ single-transmembrane protein, is a potential oncogene that promotes TNBC stemness by stabilizing EGFR. Here, we further found that SGCE depletion reduces breast cancer stem cells, partially through inhibiting the transcription of FGF-BP1, a secreted oncoprotein. Mechanistically, we demonstrate that SGCE could interact with the specific protein 1 transcription factor and translocate into the nucleus, which leads to an increase in the transcription of FGF-BP1, and the secreted FBF-BP1 activates FGF-FGFR signaling to promote cancer cell stemness. The novel SGCE-Sp1-FGF-BP1 axis provides novel potential candidate diagnostic markers and therapeutic targets for TNBC.

Src homology 3 domain binding kinase 1 protects against hepatic steatosis and insulin resistance through the Nur77-FGF21 pathway.

BACKGROUND AND AIMS: Metabolism in the liver is dysregulated in obesity, contributing to various health problems including steatosis and insulin resistance. While the pathogenesis of lipid accumulation has been extensively studied, the protective mechanism against lipid challenge in the liver remains unclear. Here, we report that Src homology 3 domain binding kinase 1 (SBK1) is a regulator of hepatic lipid metabolism and systemic insulin sensitivity in response to obesity. APPROACH AND RESULTS: Enhanced Sbk1 expression was found in the liver of high-fat diet (HFD)-induced obese mice and fatty acid (FA)-challenged hepatocytes. SBK1 knockdown in mouse liver cells augmented FA uptake and lipid accumulation. Similarly, liver-specific SBK1 knockout ( Lsko ) mice displayed more severe hepatosteatosis and higher expression of genes in FA uptake and lipogenesis than the Flox/Flox ( Fl/Fl ) control mice when fed the HFD. The HFD-fed Lsko mice also showed symptoms of hyperglycemia, poor systemic glucose tolerance, and lower insulin sensitivity than the Fl/Fl mice. On the other hand, hepatic Sbk1 overexpression alleviated the high-fructose diet-induced hepatosteatosis, hyperlipidemia, and hyperglycemia in mice. White adipose tissue browning was also observed in hepatic SBK1 -overexpressed mice. Moreover, we found that SBK1 was a positive regulator of FGF21 in the liver during energy surplus conditions. Mechanistically, SBK1 phosphorylates the orphan nuclear receptor 4A1 (Nur77) on serine 344 to promote hepatic FGF21 expression and inhibit the transcription of genes involved in lipid anabolism. CONCLUSIONS: Collectively, our data suggest that SBK1 is a regulator of the metabolic adaption against obesity through the Nur77-FGF21 pathway.

The role of TGF-ß signaling in muscle atrophy, sarcopenia and cancer cachexia.

Skeletal muscle, comprising a significant proportion (40 to 50 percent) of total body weight in humans, plays a critical role in maintaining normal physiological conditions. Muscle atrophy occurs when the rate of protein degradation exceeds protein synthesis. Sarcopenia refers to age-related muscle atrophy, while cachexia represents a more complex form of muscle wasting associated with various diseases such as cancer, heart failure, and AIDS. Recent research has highlighted the involvement of signaling pathways, including IGF1-Akt-mTOR, MuRF1-MAFbx, and FOXO, in regulating the delicate balance between muscle protein synthesis and breakdown. Myostatin, a member of the TGF-ß superfamily, negatively regulates muscle growth and promotes muscle atrophy by activating Smad2 and Smad3. It also interacts with other signaling pathways in cachexia and sarcopenia. Inhibition of myostatin has emerged as a promising therapeutic approach for sarcopenia and cachexia. Additionally, other TGF-ß family members, such as TGF-ß1, activin A, and GDF11, have been implicated in the regulation of skeletal muscle mass. Furthermore, myostatin cooperates with these family members to impair muscle differentiation and contribute to muscle loss. This review provides an overview of the significance of myostatin and other TGF-ß signaling pathway members in muscular dystrophy, sarcopenia, and cachexia. It also discusses potential novel therapeutic strategies targeting myostatin and TGF-ß signaling for the treatment of muscle atrophy.

Implications of TDP-43 in non-neuronal systems.

TAR DNA-binding protein 43 (TDP-43) is a versatile RNA/DNA-binding protein with multifaceted processes. While TDP-43 has been extensively studied in the context of degenerative diseases, recent evidence has also highlighted its crucial involvement in diverse life processes beyond neurodegeneration. Here, we mainly reviewed the function of TDP-43 in non-neurodegenerative physiological and pathological processes, including spermatogenesis, embryonic development, mammary gland development, tumor formation, and viral infection, highlighting its importance as a key regulatory factor for the maintenance of normal functions throughout life. TDP-43 exhibits diverse and sometimes opposite functionality across different cell types through various mechanisms, and its roles can shift at distinct stages within the same biological system. Consequently, TDP-43 operates in both a context-dependent and a stage-specific manner in response to a variety of internal and external stimuli. Video Abstract.

Changes in the mammary gland during aging and its links with breast diseases.

The functional capacity of organisms declines in the process of aging. In the case of breast tissue, abnormal mammary gland development can lead to dysfunction in milk secretion, a primary function, as well as the onset of various diseases, such as breast cancer. In the process of aging, the terminal duct lobular units (TDLUs) within the breast undergo gradual degeneration, while the proportion of adipose tissue in the breast continues to increase and hormonal levels in the breast change accordingly. Here, we review changes in morphology, internal structure, and cellular composition that occur in the mammary gland during aging. We also explore the emerging mechanisms of breast aging and the relationship between changes during aging and breast-related diseases, as well as potential interventions for delaying mammary gland aging and preventing breast disease.

Single-cell profiling of long noncoding RNAs and their cell lineage commitment roles via RNA-DNA-DNA triplex formation in mammary epithelium.

Long noncoding RNAs (lncRNAs), which are crucial for organ development, exhibit cell-specific expression. Thus, transcriptomic analysis based on total tissue (bulk-seq) cannot accurately reflect the expression pattern of lncRNAs. Here, we used high-throughput single-cell RNA-seq data to investigate the role of lncRNAs using the hierarchical model of mammary epithelium. With our comprehensive annotation of the mammary epithelium, lncRNAs showed much greater cell-lineage specific expression than coding genes. The lineage-specific lncRNAs were functionally correlated with lineage commitment through the coding genes via the cis- and trans-effects of lncRNAs. For the working mechanism, lncRNAs formed a triplex structure with the DNA helix to regulate downstream lineage-specific marker genes. We used lncRNA-Carmn as an example to validate the above findings. Carmn, which is specifically expressed in mammary gland stem cells (MaSCs) and basal cells, positively regulated the Wnt signaling ligand Wnt10a through formation of a lncRNA-DNA-DNA triplex, and thus controlled the stemness of MaSCs. Our study suggests that lncRNAs play essential roles in cell-lineage commitment and provides an approach to decipher lncRNA functions based on single-cell RNA-seq data.

Loss of TDP43 inhibits progression of triple-negative breast cancer in coordination with SRSF3.

Aberrant alternative splicing has been highlighted as a potential hallmark of cancer. Here, we identify TDP43 (TAR DNA-binding protein 43) as an important splicing regulator responsible for the unique splicing profile in triple-negative breast cancer (TNBC). Clinical data demonstrate that TDP43 is highly expressed in TNBC with poor prognosis. Knockdown of TDP43 inhibits tumor progression, including proliferation and metastasis, and overexpression of TDP43 promotes proliferation and malignancy of mammary epithelial cells. Deep sequencing analysis and functional experiments indicate that TDP43 alters most splicing events with splicing factor SRSF3 (serine/arginine-rich splicing factor 3), in the regulation of TNBC progression. The TDP43/SRSF3 complex controls specific splicing events, including downstream genes PAR3 and NUMB The effect of reduced metastasis and proliferation upon the knockdown of TDP43 or SRSF3 is mediated by the splicing regulation of PAR3 and NUMB exon 12, respectively. The TDP43/SRSF3 complex and downstream PAR3 isoform are potential therapeutic targets for TNBC.

RLIM is dispensable for X-chromosome inactivation in the mouse embryonic epiblast.

In female mice, two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X chromosome. Later, around implantation, epiblast cells of the inner cell mass that give rise to the embryo reactivate the paternal X chromosome and undergo a random form of XCI (rXCI). Xist, a long non-coding RNA crucial for both forms of XCI, is activated by the ubiquitin ligase RLIM (also known as Rnf12). Although RLIM is required for triggering iXCI in mice, its importance for rXCI has been controversial. Here we show that RLIM levels are downregulated in embryonic cells undergoing rXCI. Using mouse genetics we demonstrate that female cells lacking RLIM from pre-implantation stages onwards show hallmarks of XCI, including Xist clouds and H3K27me3 foci, and have full embryogenic potential. These results provide evidence that RLIM is dispensable for rXCI, indicating that in mice an RLIM-independent mechanism activates Xist in the embryo proper.

MicroRNA-1976 regulates degeneration of the sinoatrial node by targeting Cav1.2 and Cav1.3 ion channels.

Sick sinus syndrome (SSS) is primarily a disease of the elderly, and age-dependent decrease in Cav1.2 and Cav1.3 Ca2+ channels within the sinus node has been shown to play an important role in sinoatrial node (SAN) degeneration; however, posttranscriptional mechanisms regulating decrease in Cav1.2 and Cav1.3 Ca2+ channels remain unclear. Some studies have reported that microRNAs (miRNAs) are involved in age-related cardiovascular diseases. Nevertheless, little is known about the roles of miRNAs in age-related SSS. This study investigated whether miR-1976 was involved in the regulation of SAN degeneration by targeting Cav1.2 and Cav1.3 Ca2+ channels. First, using microarray-based miRNA expression profiling and qRT-PCR, we confirmed that miR-1976 was upregulated in the plasma of patients with age-related SSS relative to healthy controls. By employing target gene prediction software, luciferase assay and western blotting, we further confirmed Cav1.2 and Cav1.3 as direct targets of miR-1976. Furthermore, miR-1976 levels in rabbit SAN tissues were negatively correlated with Cav1.2 and Cav1.3 expression and intrinsic heart rates but positively correlated with corrected sinus node recovery time (CSNRT). Additionally, miR-1976 transgenic mice displayed attenuated Cav1.2 and Cav1.3 protein expression, which led to sinus node dysfunction. These results suggest that miR-1976 plays an important role in the SAN aging process by targeting Cav1.2 and Cav1.3. Thus, miR-1976 could have great potential as a noninvasive diagnostic tool and therapeutic target for SSS. These findings may reveal important insights into the pathogenesis of SSS.

Chromosome-wide gene dosage rebalance may benefit tumor progression.

The high-risk of tumor initiation in patients with Turner syndrome (TS) characterized by X chromosome monosomy in women has been well established and aneuploidy, defined as an abnormal number of chromosomes, is a common feature in human cancer. However, the underlying mechanisms of X chromosome aneuploidy promoting tumorigenesis remain obscure. We propose that chromosome-wide gene dosage imbalance (CDI) may serve as an important mechanism. Here, we assess the relative expression ratios of X chromosome and autosomes (expression ratios of X:AA) between tumor samples and adjacent normal samples across 16 tumor types using expression datasets from The Cancer Genome Atlas (TCGA) project. Our results show that the expression ratios of X:AA in tumor samples are frequently rebalanced to a lower level compared to those in adjacent normal samples, which is termed chromosome-wide gene dosage rebalance (CDR) thereafter. Gene ontology (GO) analysis of differentially expression genes from X chromosome reveals that downregulation of multicellularity-related genes and upregulation of unicellularity-related genes in tumors form a distinctive feature and enrichment analysis shows that downregulated genes are enriched in tumor suppressor genes, which indicate that CDR benefits tumor progression. Further experimental results prove that disturbance of X chromosome expression by knocking down of XIST in breast cancer cells, which functions in initiation phase of X chromosome inactivation (XCI), inhibits tumor progression. Our results demonstrate that the prevalent CDRs across tumor types serve as an important mechanism in promoting tumor progression, which partially explains the high risk of tumor in patients with TS and also provides a new cancer therapy from the CDR perspective.

Hsa_circ_0046264 up-regulated BRCA2 to suppress lung cancer through targeting hsa-miR-1245.

OBJECTIVE: Lung cancer had been leading mounts of deaths worldwide. Advances in genes microarray had helped human further understand genes and identify novel circular RNAs. This study aimed at investigating the biological functions and molecular mechanisms of hsa_circ_0046264 in lung cancer which may be helpful in lung cancer early diagnosis and clinical treatment. METHODS: Gene microarray data screened the differential gene of hsa_circ_0046264 and its downstream genes were found by bioinformatics analysis and verified by luciferase reporter assay. QRT-PCR and Western blot was used to detect the RNA and protein levels respectively. RNase R digestion confirmed the existences of circular RNA. Cell viability, invasion and apoptosis were determined by MTT assay, flow cytometry and DNA damage assay. Tumor formation in nude mice and immunohistochemistry proved the functions of hsa_circ_0046264 in vivo. RESULTS: Hsa_circ_0046264 and BRCA2 were down-regulated in lung cancer tissues while miR-1245 was up-regulated. Hsa_circ_0046264 induced apoptosis but inhibited proliferation and invasion of lung cancer cells through targeting miR-1245 to up-regulate BRCA2. Hsa_circ_0046264 inhibited the tumor growth in vivo. CONCLUSION: Hsa_circ_0046264 was a tumor suppressor in lung cancer. Overexpression of hsa_circ_0046264 could up-regulate BRCA2 expression through down-regulating of miR-1245.

Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice.

Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.

Imprinted genes: important regulators in development.

The imprinted genes are characterized for their allele-specific expression, which present a unique phenomenon in the epigenetic and developmental studies. The field had dramatically progress in terms of establishment, maintainance and function of imprinted genes in organ development. The imprinting was primarily discovered in neuro-transplantation studies, and thereafter focused on a few well-known imprinted cluster. With increasing application of omics techniques, more imprinted genes were screened and identified, which drawn great attentions and discussions by scientists in the field. One of wonderful example is peculiars extrapolation and debate of evolutional conservation of imprinted genes based on the data analysis of the whole genome DNA methylome and histone modification. In this review, the current research of imprinted genes in mammals were summarized from the feature of the genes, the roles in development, the mechanism of regulation, the advances of research methods, the evolution of parental relationship and the interaction between imprinted genes and environmental factors. This review will be helpful for general understanding and research approach of imprinted genes.

Fused in sarcoma (FUS) inhibits milk production efficiency in mammals.

Efficient milk production in mammals confers evolutionary advantages by facilitating the transmission of energy from mother to offspring. However, the regulatory mechanism responsible for the gradual establishment of milk production efficiency in mammals, from marsupials to eutherians, remains elusive. Here, we find that mammary gland of the marsupial sugar glider contained milk components during adolescence, and that mammary gland development is less dynamically cyclic compared to that in placental mammals. Furthermore, fused in sarcoma (FUS) is found to be partially responsible for this establishment of low efficiency. In mouse model, FUS inhibit mammary epithelial cell differentiation through the cyclin-dependent kinase inhibitor p57Kip2, leading to lactation failure and pup starvation. Clinically, FUS levels are negatively correlated with milk production in lactating women. Overall, our results shed light on FUS as a negative regulator of milk production, providing a potential mechanism for the establishment of milk production from marsupial to eutherian mammals.

Mcam inhibits macrophage-mediated development of mammary gland through non-canonical Wnt signaling.

While canonical Wnt signaling is well recognized for its crucial regulatory functions in cell fate decisions, the role of non-canonical Wnt signaling in adult stem cells remains elusive and contradictory. Here, we identified Mcam, a potential member of the non-canonical Wnt signaling, as an important negative regulator of mammary gland epithelial cells (MECs) by genome-scale CRISPR-Cas9 knockout (GeCKO) library screening. Loss of Mcam increases the clonogenicity and regenerative capacity of MECs, and promotes the proliferation, differentiation, and ductal morphogenesis of mammary epithelial in knockout mice. Mechanically, Mcam knockout recruits and polarizes macrophages through the Il4-Stat6 axis, thereby promoting secretion of the non-canonical Wnt ligand Wnt5a and its binding to the non-canonical Wnt signaling receptor Ryk to induce the above phenotypes. These findings reveal Mcam roles in mammary gland development by orchestrating communications between MECs and macrophages via a Wnt5a/Ryk axis, providing evidences for non-canonical Wnt signaling in mammary development.

Targeting cuproptosis by zinc pyrithione in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) poses a considerable challenge due to its aggressive nature. Notably, metal ion-induced cell death, such as ferroptosis, has garnered significant attention and demonstrated potential implications for cancer. Recently, cuproptosis, a potent cell death pathway reliant on copper, has been identified. However, whether cuproptosis can be targeted for cancer treatment remains uncertain. Here, we screened the US Food and Drug Administration (FDA)-approved drug library and identified zinc pyrithione (ZnPT) as a compound that significantly inhibited TNBC progression. RNA sequencing revealed that ZnPT disrupted copper homeostasis. Furthermore, ZnPT facilitated the oligomerization of dihydrolipoamide S-acetyltransferase, a landmark molecule of cuproptosis. Clinically, high expression levels of cuproptosis-related proteins were significantly correlated with poor prognosis in TNBC patients. Collectively, these findings indicate that ZnPT can induce cell death by targeting and disrupting copper homeostasis, providing a potential experimental foundation for exploring cuproptosis as a target in drug discovery for TNBC patients.

Insulin-like growth factor 3 regulates expression of genes encoding steroidogenic enzymes and key transcription factors in the Nile tilapia gonad.

Insulin-like growth factors (Igfs) are implicated in a wide variety of physiological roles in teleost gonadal development and reproduction. In the present study, igf3 mRNA expression in the tilapia ovary was found to be higher than in the testis from 5 to 40 days after hatching (dah) but was lower than that in testis from 50 to 70 dah. Consistently, Igf3 protein signal was detected in the somatic cells of XX and XY gonads from 10 dah until adulthood by immunohistochemistry, using a specific Igf3 polyclonal antibody. Incubation of ovarian and testicular cells in primary culture with recombinant Igf3 significantly increased nr5a1, foxl2, dmrt1, cyp19a1a, cyp11a1, cyp11b2, hsd3b2 , and cyp17a1 expression in a time- and dose-dependent manner. Promoter analysis using luciferase assays in HEK293 cells revealed that igf3 promoter activity was directly activated by Nr5a1 (Sf1) and further enhanced by Foxl2, Nr0b1a (Dax1), and Nr0b1b (Dax2) but repressed by Dmrt1 and estrogen receptor (Esr1, Esr2a, or Esr2b) along with 17beta-estradiol treatment. In addition, igf3 promoter activity was increased slightly by forskolin treatment alone but synergistically up-regulated by transfection with nr5a1. These in vitro results correlated well with the expression profile of igf3 during early gonad differentiation. Our results indicated that igf3 is involved in fish gonad steroidogenesis because of its ability to regulate the expression of foxl2, dmrt1, and nr5a1 and steroidogenic enzymes. The expression of igf3 is in turn regulated by transcription factors Foxl2, Dmrt1, and Nr5a1, as well as by 17beta-estradiol treatment.

Analysis of ancient and modern horse genomes reveals the critical impact of lncRNA-mediated epigenetic regulation on horse domestication.

Background: The domestication of horses has played critical roles in human civilizations. The excavation of ancient horse DNA provides crucial data for studying horse domestication. Studies of horse domestication can shed light on the general mechanisms of animal domestication. Objective: We wish to explore the gene transcription regulation by long noncoding RNAs (lncRNAs) that influence horse domestication. Methods: First, we assembled the ancient DNA sequences of multiple horses at different times and the genomes of horses, donkeys, and Przewalski horses. Second, we extracted sequences of lncRNA genes shared in ancient horses and sequences of lncRNA genes and the promoter regions of domestication-critical genes shared in modern horses, modern donkeys, and Przewalski horses to form two sample groups. Third, we used the LongTarget program to predict potential regulatory interactions between these lncRNAs and these domestication-critical genes and analyzed the differences between the regulation in ancient/modern horses and between horses/donkeys/Przewalski horses. Fourth, we performed functional enrichment analyses of genes that exhibit differences in epigenetic regulation. Results: First, genes associated with neural crest development and domestication syndrome are important targets of lncRNAs. Second, compared with undomesticated Przewalski horses, more lncRNAs participate in the epigenetic regulation in modern horses and donkeys, suggesting that domestication is linked to more epigenetic regulatory changes. Third, lncRNAs' potential target genes in modern horses are mainly involved in two functional areas: 1) the nervous system, behavior, and cognition, and 2) muscle, body size, cardiac function, and metabolism. Conclusion: Domestication is linked to substantial epigenetic regulatory changes. Genes associated with neural crest development and domestication syndrome underwent noticeable lncRNA-mediated epigenetic regulation changes during horse domestication.