CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning.

RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.

Unique Immune Cell Coactivators Specify Locus Control Region Function and Cell Stage.

Locus control region (LCR) functions define cellular identity and have critical roles in diseases such as cancer, although the hierarchy of structural components and associated factors that drive functionality are incompletely understood. Here we show that OCA-B, a B cell-specific coactivator essential for germinal center (GC) formation, forms a ternary complex with the lymphoid-enriched OCT2 and GC-specific MEF2B transcription factors and that this complex occupies and activates an LCR that regulates the BCL6 proto-oncogene and is uniquely required by normal and malignant GC B cells. Mechanistically, through OCA-B-MED1 interactions, this complex is required for Mediator association with the BCL6 promoter. Densely tiled CRISPRi screening indicates that only LCR segments heavily bound by this ternary complex are essential for its function. Our results demonstrate how an intimately linked complex of lineage- and stage-specific factors converges on specific and highly essential enhancer elements to drive the function of a cell-type-defining LCR.

The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Analysis of maternal genetic structure of mitochondrial DNA control region from Tai-Kadai-speaking Buyei population in southwestern China.

BACKGROUND: Even though the Buyei are a recognised ethnic group in southwestern China, there hasn't been much work done on forensic population genetics, notably using mitochondrial DNA. The sequences and haplogroups of mitochondrial DNA control regions of the Buyei peoples were studied to provide support for the establishment of a reference database for forensic DNA analysis in East Asia. METHODS AND RESULTS: The mitochondrial DNA control region sequences of 200 Buyei individuals in Guizhou were investigated. The haplotype frequencies and haplogroup distribution of the Buyei nationality in Guizhou were calculated. At the same time, the paired Fst values of the study population and other populations around the world were computed, to explore their genetic polymorphism and population relationship. A total of 179 haplotypes were detected in the Buyei population, with frequencies of 0.005-0.015. All haplotypes were assigned to 89 different haplogroups. The haplotype diversity and random matching probability were 0.999283 and 0.0063, respectively. The paired Fst genetic distances and correlation p-values among the 54 populations revealed that the Guizhou Buyei was most closely related to the Henan Han and the Guizhou Miao, and closer to the Hazara population in Pakistan and the Chiang Mai population. CONCLUSIONS: The study of mitochondrial DNA based on the maternal genetic structure of the Buyei nationality in Guizhou will benefit the establishment of an East Asian forensic DNA reference database and provide a reference for anthropological research in the future.

Identification of target cells of human papillomavirus 18 using squamocolumnar junction organoids.

Human papillomavirus 18 (HPV18) is a highly malignant HPV genotype among high-risk HPVs, characterized by the difficulty of detecting it in precancerous lesions and its high prevalence in adenocarcinomas. The cellular targets and molecular mechanisms underlying its infection remain unclear. In this study, we aimed to identify the cells targeted by HPV18 and elucidate the molecular mechanisms underlying HPV18 replication. Initially, we established a lentiviral vector (HPV18LCR-GFP vector) containing the HPV18 long control region promoter located upstream of EGFP. Subsequently, HPV18LCR-GFP vectors were transduced into patient-derived squamocolumnar junction organoids, and the presence of GFP-positive cells was evaluated. Single-cell RNA sequencing of GFP-positive and GFP-negative cells was conducted. Differentially expressed gene analysis revealed that 169 and 484 genes were significantly upregulated in GFP-positive and GFP-negative cells, respectively. Pathway analysis showed that pathways associated with cell cycle and viral carcinogenesis were upregulated in GFP-positive cells, whereas keratinization and mitophagy/autophagy-related pathways were upregulated in GFP-negative cells. siRNA-mediated luciferase reporter assay and HPV18 genome replication assay validated that, among the upregulated genes, ADNP, FHL2, and NPM3 were significantly associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. Among them, NPM3 showed substantially higher expression in HPV-related cervical adenocarcinomas than in squamous cell carcinomas, and NPM3 knockdown of HPV18-infected cells downregulated stem cell-related genes. Our new experimental model allows us to identify novel genes involved in HPV18 early promoter activities. These molecules might serve as therapeutic targets in HPV18-infected cervical lesions.

Transcriptional Regulation by (Super)Enhancers: From Discovery to Mechanisms.

Accurate control of gene expression in the right cell at the right moment is of fundamental importance to animal development and homeostasis. At the heart of gene regulation lie the enhancers, a class of gene regulatory elements that ensures precise spatiotemporal activation of gene transcription. Mammalian genomes are littered with enhancers, which are frequently organized in cooperative clusters such as locus control regions and superenhancers. Here, we discuss our current knowledge of enhancer biology, including an overview of the discovery of the various enhancer subsets and the mechanistic models used to explain their gene regulatory function.

Human papillomavirus and Merkel cell polyomavirus in Korean patients with nonsmall cell lung cancer: Evaluation and genetic variability of the noncoding control region.

Human papillomavirus (HPV) is an important causative factor of cervical cancer and is associated with nonsmall cell lung cancer (NSCLC). Merkel cell polyomavirus (MCPyV) is a rare and highly fatal cutaneous virus that can cause Merkel cell carcinoma (MCC). Although coinfection with oncogenic HPV and MCPyV may increase cancer risk, a definitive etiological link has not been established. Recently, genomic variation and genetic diversity in the MCPyV noncoding control region (NCCR) among ethnic groups has been reported. The current study aimed to provide accurate prevalence information on HPV and MCPyV infection/coinfection in NSCLC patients and to evaluate and confirm Korean MCPyV NCCR variant genotypes and sequences. DNA from 150 NSCLC tissues and 150 adjacent control tissues was assessed via polymerase chain reaction (PCR) targeting regions of the large T antigen (LT-ag), viral capsid protein 1 (VP1), and NCCR. MCPyV was detected in 22.7% (34 of 150) of NSCLC tissues and 8.0% (12 of 150) of adjacent tissues from Korean patients. The incidence rates of HPV with and without MCPyV were 26.5% (nine of 34) and 12.9% (15 of 116). The MCPyV NCCR genotype prevalence in Korean patients was 21.3% (32 of 150) for subtype I and 6% (nine of 150) for subtype IIc. Subtype I, a predominant East Asian strain containing 25 bp tandem repeats, was most common in the MCPyV NCCR data set. Our results confirm that coinfection with other tumor-associated viruses is not associated with NSCLC. Although the role of NCCR rearrangements in MCPyV infection remains unknown, future studies are warranted to determine the associations of MCPyV NCCR sequence rearrangements with specific diseases.

Establishment of COS-BK cells persistently infected with archetype BK polyomavirus.

BK polyomavirus (BKPyV) was the first human polyomavirus to be isolated from an immunosuppressed kidney transplant recipient in 1971. BKPyV reactivation causes BKPyV-associated nephropathy and hemorrhagic cystitis. However, the mechanisms underlying BKPyV replication remain unclear. In the present study, we performed the long-term cultivation of COS-7 cells transfected with archetype KOM-5 DNA, which were designated as COS-BK cells. BKPyV derived from COS-BK cells was characterized by analyzing the amount of the virus based on hemagglutination, viral replication, and the production of viral protein 1 (VP1). Immunostaining showed that VP1-positive cells accounted for a small percentage of COS-BK cells. The nucleotide sequences encompassing the origin of the DNA replication of BKPyV derived from COS-BK cells were generated from KOM-5 by the deletion of an 8-bp sequence, which did not involve T antigen binding sites. BKPyV replicated most efficiently in COS-BK cells in DMEM containing 2% fetal bovine serum. These results indicate that COS-BK cells are a suitable culture system for studying the persistent infection of archetype BKPyV.

Characterization of the mitochondrial genomes of the Mexican endemic bats Corynorhinus mexicanus and Corynorhinus leonpaniaguae (Chiroptera: Vespertilionidae).

BACKGROUND: The genus Corynorhinus is composed of four recognized species: C. rafinesquii, C. townsendii, C. mexicanus, and C. leonpaniaguae, the latter two being endemic to Mexico. According to the IUCN, C. mexicanus is considered "Near Threatened", as its populations are dwindling and habitats are affected by anthropogenic disturbance. Corynorhinus leonpaniaguae has not been assigned to an IUCN Red List risk category due to its recent description. METHODS AND RESULTS: In this study, the mitochondrial genomes of C. mexicanus and C. leonpaniaguae were assembled and characterized in detail. The mitochondrial genomes (mtDNA) of C. mexicanus and C. leonpaniaguae have lengths of 16,470 and 16,581 bp respectively, with a predominant nucleotide usage of adenine (31.670% and 31.729%, respectively) and thymine (26.15% and 26.18%, respectively). The mtDNA of C. mexicanus and C. leonpaniaguae is composed of 37 coding and non-coding elements: 22 transfer RNAs (tRNA), 13 protein-coding genes (PCGs), two ribosomal RNAs and a non-coding region, the control region, which has a length of 933 bp and 1,149 bp, respectively. All tRNAs exhibited a cloverleaf secondary structure, with the exception of trn-Ser1 which showed a deletion of the dihydrouridine arm in the two species. All PCGs are subjected to purifying selection, with atp8 being the gene showing the highest Ka/Ks value. CONCLUSIONS: These are the first whole mitogenomic resources developed for C. mexicanus and C. leonpaniaguae and enhance our knowledge of the ecology of these species and aid in their conservation.

Multiple independent origins of duplicated mitochondrial control regions indicate an apomorphy in the Thysanoptera (Insecta).

The mitochondrial genome (mitogenome) of thrips is characterized by the presence of control region (CR) duplication. However, the evolution pattern of duplicated CRs in thrips is still unclear. In this study, the multiple independent origins of duplicated CR indicated that the CR duplication was not an ancestral state for Thysanoptera. The macroevolutionary pattern suggested that the earliest CR duplication event occurred in the middle Cretaceous (94.85 Ma) coincided with rearrangement events forming the ancestors of Aeolothripidae, but much later than that forming the ancestors of the suborder Terebrantia. The mitogenome with duplicated CRs showed a higher rate of gene rearrangement. The sequence similarity of the CR copies and divergence time were negatively correlated, indicating age-related deterioration of mitochondrial function. No significant differences were found in the mitochondrial DNA, the P123 and P4FD between the single and multiple-CR charactered mitogenomes, which suggested that the duplicated CRs may not affect the replication process in thrip mitogenome. The mitogenomes with duplicated CRs (mean: 0.0088 subs/s/my) show a significantly increased evolutionary rate than that with a single one (mean: 0.0058 subs/s/my). However, it seems that this higher evolutionary rate did not have adaptive mechanisms in Terebrantia. We speculated that the duplicated CRs may cause a more intense production of energy by mitochondria, and an accelerated mutation and substitution rate is expected in such mitogenomes. Our study provided new insights into the presence of CR duplications and their evolution in the mitogenomes of thrips.

The extraordinary rearrangement of mitochondrial genome of the wheat pest, Aptinothrips stylifer and the mitochondrial phylogeny of Thripidae (Thysanoptera).

The mitochondrial gene order in Thysanoptera is notably distinct and highly rearranged, with each species exhibiting its own unique arrangement. To elucidate the relationship between gene rearrangements and phylogeny, the complete mitochondrial genome (mitogenome) of the wheat pest, Aptinothrips stylifer, was sequenced and assembled, spanning a total length of 16,033 bp. Compared with the ancestral arthropod mitogenome, significant rearrangement differences were evident in A. stylifer, whereas the gene order between A. stylifer and Anaphothrips obscurus was similar. Phylogenetic trees were reconstructed based on all 13 protein-coding gene sequences using Bayesian inference and maximum-likelihood methods, both yielding similar topological structures. Notably, A. stylifer was robustly clustered with A. obscurus, affirming its classification within Anaphothrips genus group. This exemplifies the potential correlation between gene rearrangements and phylogeny in the Thripidae family. Additionally, the mitogenome of A. stylifer exhibited several atypical features, including: (1) Three putative control regions (CRs) in close proximity, with CR2 and CR3 displaying partial similarity, and CR1 differing in base composition; (2) Two transfer RNAs (tRNAs), trnS1 and trnV, lacking the DHU arm; (3) Two ribosomal RNA (rRNA) genes inverted and positioned distant from each other; (4) Negative AT and GC skew (AT skew = -0.001, GC skew = -0.077); (5) One transposition (nad6), one inverse transposition (trnQ), four inversions (trnF, trnH, trnC, and gene block nad1-trnL1-rrnL-trnV-rrnS), and four tandem duplication random loss events; and (6) Two protein-coding genes, nad2 and atp8, terminated with an incomplete stop codon "T".

Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite.

BACKGROUND: As a population genetic tool, mitochondrial DNA is commonly divided into the ~ 1-kb control region (CR), in which single nucleotide variant (SNV) diversity is relatively high, and the coding region, in which selective constraint is greater and diversity lower, but which provides an informative phylogeny. In some species, the CR contains variable tandemly repeated sequences that are understudied due to heteroplasmy. Domestic cats (Felis catus) have a recent origin and therefore traditional CR-based analysis of populations yields only a small number of haplotypes. RESULTS: To increase resolution we used Nanopore sequencing to analyse 119 cat mitogenomes via a long-amplicon approach. This greatly improves discrimination (from 15 to 87 distinct haplotypes in our dataset) and defines a phylogeny showing similar starlike topologies within all major clades (haplogroups), likely reflecting post-domestication expansion. We sequenced RS2, a CR tandem array of 80-bp repeat units, placing RS2 array structures within the phylogeny and increasing overall haplotype diversity. Repeat number varies between 3 and 12 (median: 4) with over 30 different repeat unit types differing largely by SNVs. Five SNVs show evidence of independent recurrence within the phylogeny, and seven are involved in at least 11 instances of rapid spread along repeat arrays within haplogroups. CONCLUSIONS: In defining mitogenome variation our study provides key information for the forensic genetic analysis of cat hair evidence, and for the first time a phylogenetically informed picture of tandem repeat variation that reveals remarkably dynamic mutation processes at work in the mitochondrion.

Analysis of the complete mitogenome of Daphnia galeata from the Han River, South Korea: structure comparison and control region evolution.

Daphnia galeata is an important plankton in aquatic ecosystems. As a widely distributed species, D. galeata has been found throughout the Holarctic region. Understanding the genetic diversity and evolution of D. galeata requires the accumulation of genetic information from different locations. Even though the mitochondrial genome (mitogenome) sequence of D. galeata has already been reported, little is known about the evolution of its mitochondrial control region. In this study, D. galeata samples were collected from the Han River on the Korean Peninsula and its partial nd2 gene was sequenced for haplotype network analysis. This analysis showed that four clades of D. galeata were present in the Holarctic region. Moreover, the D. galeata examined in this study belonged to clade D and was specific to South Korea. The mitogenome of D. galeata from the Han River showed similar gene content and structure compared to sequences reported from Japan. Furthermore, the structure of control region of the Han River was similar to those of Japanese clones and differed substantially from European clone. Finally, a phylogenetic analysis based on the amino acid sequences of 13 protein-coding genes (PCGs) indicated that D. galeata from the Han River formed a cluster with clones collected from Lakes Kasumigaura, Shirakaba, and Kizaki in Japan. The differences in control region structure and stem and loop structure reflect the different evolutionary directions of the mitogenomes from Asian and European clones. These findings improve our understanding of the mitogenome structure and genetic diversity of D. galeata.

Transcriptional activity of the long control region in human papillomavirus type 33 intratype variants.

BACKGROUND: High-risk human papillomaviruses (HPVs) are responsible for the development of cervical and other anogenital cancers. Intratype sequence variants of certain high-risk HPV types (e.g. 16, 18 and 31) are thought to have different oncogenic potential, partly due to nucleotide sequence variation in the viral long control region (LCR). The LCR has an important role in the regulation of viral replication and transcription. The purpose of this study was to explore sequence variation in the LCR of HPV 33 intratype variants in Hungary and to see whether there are differences in the transcriptional activities of the variants. METHODS: The complete HPV 33 LCR was amplified from HPV 33 positive cervical samples. After sequencing the LCR variants, multiple sequence alignment and phylogenetic analyses were carried out. Representative HPV 33 LCR sequence variants were selected for cloning and functional analysis. After transient transfection of HeLa cells, luciferase reporter assays were used to analyse the transcriptional activities of different LCR variants. RESULTS: Altogether 10 different variants were identified by sequence analysis of the HPV 33 LCR. The results of phylogenetic analysis showed that 3 variants belonged to sublineage A1, while the other 7 variants clustered with sublineage A2. Variants belonging to sublineage A2 had significantly lower transcriptional activities than variants belonging to sublineage A1. Within sublineage A2, the two variants analysed had significantly different transcriptional activities, which was shown to be caused by the A7879G variation. CONCLUSIONS: Nucleotide variation in the HPV 33 LCR can result in altered transcriptional activity of the intratype variants. Our results can help to understand the correlation between LCR polymorphism and the oncogenic potential of HPV 33 variants.

Globin vector regulatory elements are active in early hematopoietic progenitor cells.

The globin genes are archetypal tissue-specific genes that are silent in most tissues but for late-stage erythroblasts upon terminal erythroid differentiation. The transcriptional activation of the ß-globin gene is under the control of proximal and distal regulatory elements located on chromosome 11p15.4, including the ß-globin locus control region (LCR). The incorporation of selected LCR elements in lentiviral vectors encoding ß and ß-like globin genes has enabled successful genetic treatment of the ß-thalassemias and sickle cell disease. However, recent occurrences of benign clonal expansions in thalassemic patients and myelodysplastic syndrome in patients with sickle cell disease call attention to the non-erythroid functions of these powerful vectors. Here we demonstrate that lentivirally encoded LCR elements, in particular HS1 and HS2, can be activated in early hematopoietic cells including hematopoietic stem cells and myeloid progenitors. This activity is position-dependent and results in the transcriptional activation of a nearby reporter gene in these progenitor cell populations. We further show that flanking a globin vector with an insulator can effectively restrain this non-erythroid activity without impairing therapeutic globin expression. Globin lentiviral vectors harboring powerful LCR HS elements may thus expose to the risk of trans-activating cancer-related genes, which can be mitigated by a suitable insulator.

Haplotype study of the mitochondrial control region of broiler breeds with different growth rates.

This study discussed the haplotype characteristics of mitochondrial control region (D-loop region) of broiler breeds with different growth rates as well as the relationships between different haplotypes and growth performance. The D-loop region's sequence size of the 974 individuals from 20 broiler breeds ranged from 1,231 to 1,232bp, as a C base deletion at 859 bp. A total of 52 mutation sites and 72 haplotypes were detected, which were divided into four haplogroups, A, B, C and E. Of these, haplogroup E was the dominant haplogroup among 817 broilers and all medium/fast-growing broiler breeds. While its proportion was not exceed 40.00% among others types breeds. Correlation analysis showed that there was a significant negative correlation between birth weight and haplogroup A and a significant positive correlation between birth weight and haplogroup E. Age in days and feed conversion ratios were positively correlated with haplogroup B but negatively correlated with haplogroup E when the average body weight was approximately 1.8 kg. The cluster analysis showed that haplogroups A and B with Gallus gallus spadiceus and E with Gallus gallus murgha, respectively clustered into one branch. This study provided a theoretical basis for broiler breeding and genetic resource protection, development and utilization.

Mitogenomics of the suborder Cottoidei (Teleostei: Perciformes): Improved assemblies, mitogenome features, phylogeny, and ecological implications.

We determined the mitogenome of Cyclopterus lumpus using a hybrid sequencing approach, and another four closely related species in the Liparidae based on available next-generation sequence data. We found that the mitogenome of C. lumpus was 17,266 bp in length, where the length and organisation were comparable to those reported for cottoids. However, we found a GC-homopolymer region in the intergenic space between tRNALeu2 and ND1 in liparids and cyclopterids. Phylogenetic reconstruction confirmed the monophyly of infraorders and firmly supported a sister-group relationship between Cyclopteridae and Liparidae. Purifying selection was the predominant force in the evolution of cottoid mitogenomes. There was significant evidence of relaxed selective pressures along the lineage of deep-sea fish, while selection was intensified in the freshwater lineage. Overall, our analysis provides a necessary expansion in the availability of mitogenomic sequences and sheds light on mitogenomic adaptation in Cottoidei fish inhabiting different aquatic environments.

Population structure of mud flounder Paralichthys orbignyanus from the south-western Atlantic Ocean.

The mud flounder Paralichthys orbignyanus (Pleuronectiformes, Paralichthyidae) inhabits shallow waters of low salinities and mud bottoms in the temperate marine coastal regions of the Bonaerensean Ecoregion of the Argentinean Biogeographic Province in the south-western Atlantic Ocean. Specimens of P. orbignyanus were collected from Lagoa dos Patos (LDP) (southern Brazil), Mar Chiquita (MCH) and Marisol (MAR) both located in Buenos Aires (Argentina), and San Antonio Oeste (SAO) in the San Matías Gulf, Rio Negro (Argentina). A fragment of the mitochondrial DNA of the Control Region and seven microsatellite loci were characterized. In the Control Region, P. orbignyanus showed high variability, low nucleotide diversity, mild population expansion and a coalescence time of 35,000 years before the present. Flounders provided evidence of a genetic structure between the sampling sites LDP, MCH, MAR vs. SAO. On the other hand, P. orbignyanus displayed a lower to moderate contemporary genetic structure among all samples except between LDP and MCH. With no evidence of isolation by distance, this analysis supports a model of limited gene flow that is likely to be associated with a consistent larvae retention in all sampling sites. In addition, the present connectivity is ascribed to a lower migration process from SAO in the San Matías Gulf congruent with the prevailing littoral drift.

Genetic Characteristics of the Baikal Seal Pusa sibirica Population.

Nucleotide sequence diversity in two mtDNA loci (the cytochrome b gene and the control region) was for the first time studied in the Baikal seal Pusa sibirica with the use of several spatiotemporal samples. The population was found to be evolutionarily young and to be in the stage of demographic expansion.

Characterization of the mitochondrial genomes of Bradysia hygida, Phytosciara flavipes and Trichosia splendens (Diptera: Sciaridae) and novel insights on the control region of sciarid mitogenomes.

Sciarids, also called "fungus gnats" are small, almost entirely dark-coloured insects. Sciarid larvae feed on different substrates and can infest agricultural crops and mushroom nurseries, causing economic losses. Of the 2174 Diptera mitogenome sequences currently available in GenBank, only eight are from the Sciaridae family, none of which are complete circular molecules. Here we describe the mitogenome sequences of three sciarid species: Phytosciara flavipes, Trichosia splendens and Bradysia hygida and provide novel insights on the control region of sciarid mitogenomes. The assembled mitogenomes range from 16,062 bp in P. flavipes to 17,095 bp in B. hygida. All 13 protein coding genes, 22 tRNAs and 2 rRNAs characteristic of insect mitogenomes were identified, but the sequence of the control region could not be determined. Experimental results suggest that the B. hygida control region is about 21 kb long resulting in a 37 kb long mitogenome which constitutes the largest insect mitochondrial genome described so far. Phylogenetic analysis using all Bibionomorpha mitogenome sequences available in GenBank strongly supports the Sciaridae monophyly and led to the identification of species and subfamily specific gene rearrangements. Our study extends the knowledge of this large and diverse insect family that includes agricultural pest species.