A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis.

The long interspersed element 1 (LINE-1 or L1) integration is affected by many cellular factors through various mechanisms. Some of these factors are required for L1 amplification, while others either suppress or enhance specific steps during L1 propagation. Previously, TRIM28 has been identified to suppress transposable elements, including L1 expression via its canonical role in chromatin remodeling. Here, we report that TRIM28 through its B box domain increases L1 retrotransposition and facilitates shorter cDNA and L1 insert generation in cultured cells. Consistent with the latter, we observe that tumor specific L1 inserts are shorter in endometrial, ovarian, and prostate tumors with higher TRIM28 mRNA expression than in those with lower TRIM28 expression. We determine that three amino acids in the B box domain that are involved in TRIM28 multimerization are critical for its effect on both L1 retrotransposition and cDNA synthesis. We provide evidence that B boxes from the other two members in the Class VI TRIM proteins, TRIM24 and TRIM33, also increase L1 retrotransposition. Our findings could lead to a better understanding of the host/L1 evolutionary arms race in the germline and their interplay during tumorigenesis.

SCIFER: approach for analysis of LINE-1 mRNA expression in single cells at a single locus resolution.

BACKGROUND: Endogenous expression of L1 mRNA is the first step in an L1-initiated mutagenesis event. However, the contribution of individual cell types to patterns of organ-specific L1 mRNA expression remains poorly understood, especially at single-locus resolution. We introduce a method to quantify expression of mobile elements at the single-locus resolution in scRNA-Seq datasets called Single Cell Implementation to Find Expressed Retrotransposons (SCIFER). SCIFER aligns scRNA-Seq reads uniquely to the genome and extracts alignments from single cells by cell-specific barcodes. In contrast to the alignment performed using default parameters, this alignment strategy increases accuracy of L1 locus identification by retaining only reads that are uniquely mapped to individual L1 loci. L1 loci expressed in single cells are unambiguously identified using a list of L1 loci manually validated to be expressed in bulk RNA-Seq datasets generated from the same cell line or organ. RESULTS: Validation of SCIFER using MCF7 cells determined technical parameters needed for optimal detection of L1 expression in single cells. We show that unsupervised analysis of L1 expression in single cells exponentially inflates both the levels of L1 expression and the number of expressed L1 loci. Application of SCIFER to analysis of scRNA-Seq datasets generated from mouse and human testes identified that mouse Round Spermatids and human Spermatogonia, Spermatocytes, and Round Spermatids express the highest levels of L1 mRNA. Our analysis also determined that similar to mice, human testes from unrelated individuals share as much as 80% of expressed L1 loci. Additionally, SCIFER determined that individual mouse cells co-express different L1 sub-families and different families of transposable elements, experimentally validating their co-existence in the same cell. CONCLUSIONS: SCIFER detects mRNA expression of individual L1 loci in single cells. It is compatible with scRNA-Seq datasets prepared using traditional sequencing methods. Validated using a human cancer cell line, SCIFER analysis of mouse and human testes identified key cell types supporting L1 expression in these species. This will further our understanding of differences and similarities in endogenous L1 mRNA expression patterns in mice and humans.

Analysis of epigenetic features characteristic of L1 loci expressed in human cells.

Only a select few L1 loci in the human genome are expressed in any given cell line or organ, likely to minimize damage done to the genome. The epigenetic features and requirements of expressed L1 loci are currently unknown. Using human cells and comprehensive epigenetic analysis of individual expressed and unexpressed L1 loci, we determined that endogenous L1 transcription depends on a combination of epigenetic factors, including open chromatin, activating histone modifications, and hypomethylation at the L1 promoter. We demonstrate that the L1 promoter seems to require interaction with enhancer elements for optimal function. We utilize epigenetic context to predict the expression status of L1Hs loci that are poorly mappable with RNA-Seq. Our analysis identified a population of 'transitional' L1 loci that likely have greater potential to be activated during the epigenetic dysregulation seen in tumors and during aging because they are the most responsive to targeted CRISPR-mediated delivery of trans-activating domains. We demonstrate that an engineered increase in endogenous L1 mRNA expression increases Alu mobilization. Overall, our findings present the first global and comprehensive analysis of epigenetic status of individual L1 loci based on their expression status and demonstrate the importance of epigenetic context for L1 expression heterogeneity.

A Drosophila insulator interacting protein suppresses enhancer-blocking function and modulates replication timing.

Insulators play important roles in genome structure and function in eukaryotes. Interactions between a DNA binding insulator protein and its interacting partner proteins define the properties of each insulator site. The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. The Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the gypsy retrotransposon. Insulator activity relies on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulator complexes is unclear. Here, we demonstrate that HIPP1 colocalizes with the Su(Hw) insulator complex in polytene chromatin and in stress-induced insulator bodies. We find that the overexpression of either HIPP1 or Su(Hw) or mutation of the HIPP1 crotonase-like domain (CLD) causes defects in cell proliferation by limiting the progression of DNA replication. We also show that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function, while mutation of the HIPP1 CLD does not affect Su(Hw) enhancer blocking. These findings demonstrate a functional relationship between HIPP1 and the Su(Hw) insulator complex and suggest that the CLD, while not involved in enhancer blocking, influences cell cycle progression.

Organ-, sex- and age-dependent patterns of endogenous L1 mRNA expression at a single locus resolution.

Expression of L1 mRNA, the first step in the L1 copy-and-paste amplification cycle, is a prerequisite for L1-associated genomic instability. We used a reported stringent bioinformatics method to parse L1 mRNA transcripts and measure the level of L1 mRNA expressed in mouse and rat organs at a locus-specific resolution. This analysis determined that mRNA expression of L1 loci in rodents exhibits striking organ specificity with less than 0.8% of loci shared between organs of the same organism. This organ specificity in L1 mRNA expression is preserved in male and female mice and across age groups. We discovered notable differences in L1 mRNA expression between sexes with only 5% of expressed L1 loci shared between male and female mice. Moreover, we report that the levels of total L1 mRNA expression and the number and spectrum of expressed L1 loci fluctuate with age as independent variables, demonstrating different patterns in different organs and sexes. Overall, our comparisons between organs and sexes and across ages ranging from 2 to 22 months establish previously unforeseen dynamic changes in L1 mRNA expression in vivo. These findings establish the beginning of an atlas of endogenous L1 mRNA expression across a broad range of biological variables that will guide future studies.

Mutations in the insulator protein Suppressor of Hairy wing induce genome instability.

Insulator proteins orchestrate the three-dimensional organization of the genome. Insulators function by facilitating communications between regulatory sequences and gene promoters, allowing accurate gene transcription regulation during embryo development and cell differentiation. However, the role of insulator proteins beyond genome organization and transcription regulation remains unclear. Suppressor of Hairy wing [Su(Hw)] is a Drosophila insulator protein that plays an important function in female oogenesis. Here we find that su(Hw) has an unsuspected role in genome stability during cell differentiation. We show that su(Hw) mutant developing egg chambers have poorly formed microtubule organization centers (MTOCs) in the germarium and display mislocalization of the anterior/posterior axis specification factor gurken in later oogenesis stages. Additionally, eggshells from partially rescued su(Hw) mutant female germline exhibit dorsoventral patterning defects. These phenotypes are very similar to phenotypes found in the important class of spindle mutants or in piRNA pathway mutants in Drosophila, in which defects generally result from the failure of germ cells to repair DNA damage. Similarities between mutations in su(Hw) and spindle and piRNA mutants are further supported by an excess of DNA damage in nurse cells, and because Gurken localization defects are partially rescued by mutations in the ATR (mei-41) and Chk1 (grapes) DNA damage response genes. Finally, we also show that su(Hw) mutants produce an elevated number of chromosome breaks in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) is necessary for the maintenance of genome integrity during Drosophila development, in both germline and dividing somatic cells.