A long transcript mutant of the rubisco activase gene RCA upregulated by the transcription factor Ghd2 enhances drought tolerance in rice.

The transcription factor Ghd2 increases rice yield potential under normal conditions and accelerates leaf senescence under drought stress. However, its mechanism on the regulation of leaf senescence under drought stress remains unclear. In the present study, to unveil the mechanism, one target of Ghd2, the Rubisco activase gene RCA, was identified through the combined analysis of Ghd2-CRISPR transcriptome data and Ghd2-overexpression microarray data. Ghd2 binds to the 'CACA' motif in the RCA promoter by its CCT domain and upregulates RCA expression. RCA has alternative transcripts, RCAS and RCAL, which are predominantly expressed under normal conditions and drought stress, respectively. Similar to Ghd2-overexpressing plants, RCAL-overexpressing plants were more sensitive to drought stress than the wild-type. However, the plants overexpressing RCAS showed a weak drought-sensitive phenotype. Moreover, RCAL knockdown and knockout plants did not show yield loss under normal conditions, but exhibited enhanced drought tolerance and delayed leaf senescence. The chlorophyll content, the free amino acid content and the expression of senescence-related genes in the RCAL mutant were lower than those in the wild-type plants under drought stress. In summary, Ghd2 induces leaf senescence by upregulating RCAL expression under drought stress, and the RCAL mutant has important values in breeding drought-tolerant varieties.

Interleukin-22 protects from endotoxemia by inducing suppressive F4/80+Ly6GhiLy6Chi cells population.

BACKGROUND: Excessive inflammatory response is the primary cause of early death in patients with endotoxemia. Interleukin 22 (IL-22) has been shown to play critical roles in the modulation of infectious diseases, but its function in regulating immune responses during endotoxemia remains unclear. METHODS: Lipopolysaccharide (LPS) was used to induce endotoxemia mouse model with or without a recombinant fusion protein containing human IL-22 (F-652). IL-6, TNF-α, IL-1ß, and MCP-1 were measured by ELISA assays. The type of macrophage was assessed by flow cytometry. Real-time PCR was used to detect the expression of S100A9. RESULTS: We found that F-652 injection significantly improved the survival rates and reduced pro-inflammatory cytokines (IL-6, TNF-a, IL-1ß, MCP-1) in LPS-induced endotoxemia mice. However, the mice injected with F-652 had a higher number of infiltrated immune cells after LPS treatment, suggesting an impaired immune response. Flow cytometry analysis showed a higher number of F4/80+Ly6GhiLy6Chi cells that highly expressed M2-like macrophage markers (Ym1, Arg, CCL17) in the peritoneal cavity of the F-652-treated endotoxemia mice. Further investigation found that these suppressive M2 macrophages might be induced by F-652 since the F-652 treatment could increase S100A9 in vitro. CONCLUSIONS: Our study suggests that IL-22 has a protective role against endotoxemia by inducing the development of immunosuppressive cells through S100A9.

cfDNApipe: a comprehensive quality control and analysis pipeline for cell-free DNA high-throughput sequencing data.

MOTIVATION: Cell-free DNA (cfDNA) is gaining substantial attention from both biological and clinical fields as a promising marker for liquid biopsy. Many aspects of disease-related features have been discovered from cfDNA high-throughput sequencing (HTS) data. However, there is still a lack of integrative and systematic tools for cfDNA HTS data analysis and quality control (QC). RESULTS: Here, we propose cfDNApipe, an easy-to-use and systematic python package for cfDNA whole-genome sequencing (WGS) and whole-genome bisulfite sequencing (WGBS) data analysis. It covers the entire analysis pipeline for the cfDNA data, including raw sequencing data processing, QC and sophisticated statistical analysis such as detecting copy number variations (CNVs), differentially methylated regions and DNA fragment size alterations. cfDNApipe provides one-command-line-execution pipelines and flexible application programming interfaces for customized analysis. AVAILABILITY AND IMPLEMENTATION: https://xwanglabthu.github.io/cfDNApipe/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

OsTMF attenuates cold tolerance by affecting cell wall properties in rice.

Plant cell wall composition and structure can be modified as plants adapt to environmental stresses; however, the underlying regulatory mechanisms remain elusive. Here, we report that OsTMF, a homologue of the human TATA modulatory factor (TMF) in rice (Oryza sativa) and highly conserved in plants, negatively regulates cold tolerance through modification of cell wall properties. Cold stress increased the expression of OsTMF and accumulation of OsTMF in the nucleus, where OsTMF acts as a transcription activator and modulates the expression of genes involved in pectin degradation (OsBURP16), cellulose biosynthesis (OsCesA4 and OsCesA9), and cell wall structural maintenance (genes encoding proline-rich proteins and peroxidases). OsTMF directly activated the expression of OsBURP16, OsCesA4, and OsCesA9 through binding to the TATA cis-elements in their promoters. Under cold stress conditions, OsTMF negatively regulated pectin content and peroxidase activity and positively regulated cellulose content, causing corresponding alterations to cell wall properties, all of which collectively contribute to the negative effect of OsTMF on cold tolerance. Our findings unravel a previously unreported molecular mechanism of a conserved plant TMF protein in the regulation of cell wall changes under cold stress.

LncRNA HOTAIR promotes endometrial fibrosis by activating TGF-ß1/Smad pathway.

Homeobox transcript antisense RNA (HOTAIR) is a long non-coding RNA associated with a number of fibrosis-related diseases. The aim of this study was to investigate the specific role of HOTAIR in the development of endometrial fibrosis and to identify the molecular mechanisms underlying this process. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression levels of HOTAIR in samples of intrauterine adhesion (IUA) tissue and in endometrial stromal cells (ESCs) that had been treated with transforming growth factor beta 1 (TGF-ß1). Additionally, we transfected ESCs with either overexpression plasmid (pcDNA-HOTAIR) or silencing construct (si-HOTAIR) and then treated these cells with TGF-ß1. We then performed RT-qPCR and western blot analysis, along with cell proliferation and apoptosis assays, to investigate the effects of HOTAIR on the transdifferentiation of ESCs into myofibroblasts. The results showed that the expression levels of HOTAIR were significantly elevated in IUA tissue and in ESCs that had been treated with TGF-ß1. The overexpression of HOTAIR had a pro-fibrotic effect on ESCs, while the silencing of HOTAIR exerted an anti-fibrotic effect. Most importantly, the protein expression levels of p-Smad2 and p-Smad3 were significantly upregulated in TGF-ß1-treated ESCs transfected with pcDNA-HOTAIR and were downregulated after transfection with si-HOTAIR constructs. These data indicate that HOTAIR promotes endometrial fibrosis by activating the TGF-ß1/Smad signaling pathway, suggesting that the inhibition of HOTAIR may represent a promising therapeutic option for suppressing endometrial fibrosis.

Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels.

The transcription factor far upstream element binding protein (FBP) binds and activates the MYC promoter when far upstream element is via TFIIH helicase activity early in the transcription cycle. The fundamental biology and pathology of FBP are complex. In some tumors FBP seems pro-oncogenic, whereas in others it is a tumor suppressor. We generated an FBP knockout (Fubp1(-/-)) mouse to study FBP deficiency. FBP is embryo lethal from embryonic day 10.5 to birth. A spectrum of pathology is associated with FBP loss; besides cerebral hyperplasia and pulmonary hypoplasia, pale livers, hypoplastic spleen, thymus, and bone marrow, cardiac hypertrophy, placental distress, and small size were all indicative of anemia. Immunophenotyping of hematopoietic cells in wild-type versus knockout livers revealed irregular trilineage anemia, with deficits in colony formation. Despite normal numbers of hematopoietic stem cells, transplantation of Fubp1(-/-) hematopoietic stem cells into irradiated mice entirely failed to reconstitute hematopoiesis. In competitive transplantation assays against wild-type donor bone marrow, Fubp1(-/-) hematopoietic stem cells functioned only sporadically at a low level. Although cultures of wild-type mouse embryo fibroblasts set Myc levels precisely, Myc levels of mouse varied wildly between fibroblasts harvested from different Fubp1(-/-) embryos, suggesting that FBP contributes to Myc set point fixation. FBP helps to hold multiple physiologic processes to close tolerances, at least in part by constraining Myc expression.

Ghd2, a CONSTANS-like gene, confers drought sensitivity through regulation of senescence in rice.

CONSTANS (CO)-like genes have been intensively investigated for their roles in the regulation of photoperiodic flowering, but very limited information has been reported on their functions in other biological processes. Here, we found that a CO-like gene, Ghd2 (Grain number, plant height, and heading date2), which can increase the yield potential under normal growth condition just like its homologue Ghd7, is involved in the regulation of leaf senescence and drought resistance. Ghd2 is expressed mainly in the rice (Oryza sativa) leaf with the highest level detected at the grain-filling stage, and it is down-regulated by drought stress conditions. Overexpression of Ghd2 resulted in significantly reduced drought resistance, while its knockout mutant showed the opposite phenotype. The earlier senescence symptoms and the transcript up-regulation of many senescence-associated genes (SAGs) in Ghd2-overexpressing transgenic rice plants under drought stress conditions indicate that Ghd2 plays essential roles in accelerating drought-induced leaf senescence in rice. Moreover, developmental and dark-induced leaf senescence was accelerated in the Ghd2-overexpressing rice and delayed in the ghd2 mutant. Several SAGs were confirmed to be regulated by Ghd2 using a transient expression system in rice protoplasts. Ghd2 interacted with several regulatory proteins, including OsARID3, OsPURα, and three 14-3-3 proteins. OsARID3 and OsPURα showed expression patterns similar to Ghd2 in rice leaves, with the highest levels at the grain-filling stage, whereas OsARID3 and the 14-3-3 genes responded differently to drought stress conditions. These results indicate that Ghd2 functions as a regulator by integrating environmental signals with the senescence process into a developmental programme through interaction with different proteins.

JTV1 co-activates FBP to induce USP29 transcription and stabilize p53 in response to oxidative stress.

c-myc and p53 networks control proliferation, differentiation, and apoptosis and are responsive to, and cross-regulate a variety of stresses and metabolic and biosynthetic processes. At c-myc, the far upstream element binding protein (FBP) and FBP-interacting repressor (FIR) program transcription by looping to RNA polymerase II complexes engaged at the promoter. Another FBP partner, JTV1/AIMP2, a structural subunit of a multi-aminoacyl-tRNA synthetase (ARS) complex, has also been reported to stabilize p53 via an apparently independent mechanism. Here, we show that in response to oxidative stress, JTV1 dissociates from the ARS complex, translocates to the nucleus, associates with FBP and co-activates the transcription of a new FBP target, ubiquitin-specific peptidase 29 (USP29). A previously uncharacterized deubiquitinating enzyme, USP29 binds to, cleaves poly-ubiquitin chains from, and stabilizes p53. The accumulated p53 quickly induces apoptosis. Thus, FBP and JTV1 help to coordinate the molecular and cellular response to oxidative stress.

Natural uORF variation in plants.

Taking advantage of natural variation promotes our understanding of phenotypic diversity and trait evolution, ultimately accelerating plant breeding, in which the identification of causal variations is critical. To date, sequence variations in the coding region and transcription level polymorphisms caused by variations in the promoter have been prioritized. An upstream open reading frame (uORF) in the 5' untranslated region (5' UTR) regulates gene expression at the post-transcription or translation level. In recent years, studies have demonstrated that natural uORF variations shape phenotypic diversity. This opinion article highlights recent researches and speculates on future directions for natural uORF variation in plants.

Use of 6-minute walk test for assessing severity of interstitial lung disease: an observational study.

BACKGROUND: The 6-minute walk test (6MWT) is a potential tool for assessing the severity of interstitial lung disease (ILD). OBJECTIVES: To explore the relationship between 6MWT results and traditional measures including pulmonary function and chest computed tomography(CT) and to determine factors that might influence the 6-minute walk distance (6MWD). METHODS: Seventy-three patients with ILD were enrolled at Peking University First Hospital. All patients underwent 6MWT, pulmonary CT, and pulmonary function tests and their correlations were analyzed. Multivariate regression analysis was used to identify factors that might impact 6MWD.  Results: Thirty (41.4%) of the patients were female and the mean age was 66.1 ± 9.6 years. 6MWD was correlated with forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), total lung capacity (TLC), diffusing capacity for carbon monoxide (DLCO) and DLCO%pred. The decrease in oxygen saturation (SpO2) after the test was correlated with FEV1%pred, FVC%pred, TLC, TLC%pred, DLCO, DLCO%pred and the percentage of normal lung calculated by quantitative CT. The increase in Borg dyspnea scale was correlated with FEV1, DLCO and the percentage of normal lung. The backward multivariate model (F = 15.257, P < 0.001, adjusted R2 = 0.498) indicated that 6MWD was predicted by age, height, body weight, increase in heart rate, and DLCO. CONCLUSIONS: The 6MWT results were closely correlated with pulmonary function and quantitative CT in patients with ILD. However, in addition to disease severity, 6MWD was also influenced by individual characteristics and the degree of patient effort, which should thus be considered by clinicians when interpreting 6WMT results.

The CCT transcriptional activator Ghd2 constantly delays the heading date by upregulating CO3 in rice.

CONSTANS, CO-like, and TOC1 (CCT) family genes play important roles in regulating heading date, which exerts a large impact on the regional and seasonal adaptation of rice. Previous studies have shown that Grain number, plant height, and heading date2 (Ghd2) exhibit a negative response to drought stress by directly upregulating Rubisco activase and exerting a negative effect on heading date. However, the target gene of Ghd2 regulating heading date is still unknown. In this study, CO3 is identified by analyzing ChIP-seq data. Ghd2 activates CO3 expression by binding to the CO3 promoter through its CCT domain. EMSA experiments show that the motif CCACTA in the CO3 promoter was recognized by Ghd2. A comparison of the heading dates among plants with CO3 knocked out or overexpressed and double mutants overexpressing Ghd2 with CO3 knocked out shows that CO3 negatively and constantly regulates flowering by repressing the transcription of Ehd1, Hd3a, and RFT1. In addition, the target genes of CO3 are explored via a comprehensive analysis of DAP-seq data and RNA-seq data. Taken together, these results suggest that Ghd2 directly binds to the downstream gene CO3, and the Ghd2-CO3 module constantly delays heading date via the Ehd1-mediated pathway.

Hierarchical mechanisms build the DNA-binding specificity of FUSE binding protein.

The far upstream element (FUSE) binding protein (FBP), a single-stranded nucleic acid binding protein, is recruited to the c-myc promoter after melting of FUSE by transcriptionally generated dynamic supercoils. Via interactions with TFIIH and FBP-interacting repressor (FIR), FBP modulates c-myc transcription. Here, we investigate the contributions of FBP's 4 K Homology (KH) domains to sequence selectivity. EMSA and missing contact point analysis revealed that FBP contacts 4 separate patches spanning a large segment of FUSE. A SELEX procedure using paired KH-domains defined the preferred subsequences for each KH domain. Unexpectedly, there was also a strong selection for the noncontacted residues between these subsequences, showing that the contact points must be optimally presented in a backbone that minimizes secondary structure. Strategic mutation of contact points defined in this study disabled FUSE activity in vivo. Because the biological specificity of FBP is tuned at several layers: (i) accessibility of the site; (ii) supercoil-driven melting; (iii) presentation of unhindered bases for recognition; and (iv) modular interaction of KH-domains with cognate bases, the FBP-FIR system and sequence-specific, single-strand DNA binding proteins in general are likely to prove versatile tools for adjusting gene expression.

FBPs are calibrated molecular tools to adjust gene expression.

The three far-upstream element (FUSE) binding protein (FBP) family members have been ascribed different functions in gene regulation. They were therefore examined with various biochemical, molecular biological, and cell biological tests to evaluate whether their sequence differences reflect functional customization or neutral changes at unselected residues. Each FBP displayed a characteristic profile of intrinsic transcription activation and repression, binding with protein partners, and subcellular trafficking. Although some differences, such as weakened FBP3 nuclear localization, were predictable from primary sequence differences, the unexpected failure of FBP3 to bind the FBP-interacting repressor (FIR) was traced to seemingly conservative substitutions within a small patch of an N-terminal alpha-helix. The transactivation strength and the FIR-binding strength of the FBPs were in the opposite order. Despite their distinguishing features and differential activities, the FBPs traffic to shared subnuclear sites and regulate many common target genes, including c-myc. Though a variety of functions have been attributed to the FBPs, based upon their panel of shared and unique features, we propose that they constitute a molecular regulatory kit that tunes the expression of shared targets through a common mechanism.

The dynamic response of upstream DNA to transcription-generated torsional stress.

The torsional stress caused by counter-rotation of the transcription machinery and template generates supercoils in a closed topological domain, but has been presumed to be too short-lived to be significant in an open domain. This report shows that transcribing RNA polymerases dynamically sustain sufficient torsion to perturb DNA structure even on linear templates. Assays to capture and measure transcriptionally generated torque and to trap short-lived perturbations in DNA structure and conformation showed that the transient forces upstream of active promoters are large enough to drive the supercoil-sensitive far upstream element (FUSE) of the human c-myc into single-stranded DNA. An alternative non-B conformation of FUSE found in stably supercoiled DNA is not accessible dynamically. These results demonstrate that dynamic disturbance of DNA structure provides a real-time measure of ongoing genetic activity.

TFIIH operates through an expanded proximal promoter to fine-tune c-myc expression.

A continuous stream of activating and repressing signals is processed by the transcription complex paused at the promoter of the c-myc proto-oncogene. The general transcription factor IIH (TFIIH) is held at promoters prior to promoter escape and so is well situated to channel the input of activators and repressors to modulate c-myc expression. We have compared cells expressing only a mutated p89 (xeroderma pigmentosum complementation group B [XPB]), the largest TFIIH subunit, with the same cells functionally complemented with the wild-type protein (XPB/wt-p89). Here, we show structural, compositional, and functional differences in transcription complexes between XPB and XPB/wt-89 cells at the native c-myc promoter. Remarkably, although the mean levels of c-Myc are only modestly elevated in XPB compared to those in XPB/wt-p89 cells, the range of expression and the cell-to-cell variation of c-Myc are markedly increased. Our modeling indicates that the data can be explained if TFIIH integrates inputs from multiple signals, regulating transcription at multiple kinetically equivalent steps between initiation and promoter escape. This helps to suppress the intrinsic noise of transcription and to ensure the steady transcriptional output of c-myc necessary for cellular homeostasis.

Translational repression by a miniature inverted-repeat transposable element in the 3' untranslated region.

Transposable elements constitute a substantial portion of eukaryotic genomes and contribute to genomic variation, function, and evolution. Miniature inverted-repeat transposable elements (MITEs), as DNA transposons, are widely distributed in plant and animal genomes. Previous studies have suggested that retrotransposons act as translational regulators; however, it remains unknown how host mRNAs are influenced by DNA transposons. Here we report a translational repression mechanism mediated by a stowaway-like MITE (sMITE) embedded in the 3'-untranslated region (3'-UTR) of Ghd2, a member of the CCT (CONSTANS [CO], CO-LIKE and TIMING OF CAB1) gene family in rice. Ghd2 regulates important agronomic traits, including grain number, plant height and heading date. Interestingly, the translational repression of Ghd2 by the sMITE mainly relies on Dicer-like 3a (OsDCL3a). Furthermore, other MITEs in the 3'-UTRs of different rice genes exhibit a similar effect on translational repression, thus suggesting that MITEs may exert a general regulatory function at the translational level.

Transcription-dependent dynamic supercoiling is a short-range genomic force.

Transcription has the capacity to mechanically modify DNA topology, DNA structure and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dynamics, we charted an ENCODE map of transcription-dependent dynamic supercoiling in human Burkitt's lymphoma cells by using psoralen photobinding to probe DNA topology in vivo. Dynamic supercoils spread ~1.5 kilobases upstream of the start sites of active genes. Low- and high-output promoters handled this torsional stress differently, as shown by using inhibitors of transcription and topoisomerases and by chromatin immunoprecipation of RNA polymerase and topoisomerases I and II. Whereas lower outputs are managed adequately by topoisomerase I, high-output promoters additionally require topoisomerase II. The genome-wide coupling between transcription and DNA topology emphasizes the importance of dynamic supercoiling for gene regulation.

Interactions between SAP155 and FUSE-binding protein-interacting repressor bridges c-Myc and P27Kip1 expression.

Oncogenic c-Myc plays a critical role in cell proliferation, apoptosis, and tumorigenesis, but the precise mechanisms that drive this activity remain largely unknown. P27Kip1 (CDKN1B) arrests cells in G1, and SAP155 (SF3B1), a subunit of the essential splicing factor 3b (SF3b) subcomplex of the spliceosome, is required for proper P27 pre-mRNA splicing. FUSE-binding protein-interacting repressor (FIR), a splicing variant of PUF60 lacking exon5, is a c-Myc transcriptional target that suppresses the DNA helicase p89 (ERCC3) and is alternatively spliced in colorectal cancer lacking the transcriptional repression domain within exon 2 (FIRΔexon2). FIR and FIRΔexon2 form a homo- or hetero-dimer that complexes with SAP155. Our study indicates that the FIR/FIRΔexon2/SAP155 interaction bridges c-Myc and P27 expression. Knockdown of FIR/FIRΔexon2 or SAP155 reduced p27 expression, inhibited its pre-mRNA splicing, and reduced CDK2/Cyclin E expression. Moreover, spliceostatin A, a natural SF3b inhibitor, markedly inhibited P27 expression by disrupting its pre-mRNA splicing and reduced CDK2/Cyclin E expression. The expression of P89, another FIR target, was increased in excised human colorectal cancer tissues. Knockdown of FIR reduced P89; however, the effects on P27 and P89 expression are not simply or directly related to altered FIR expression levels, indicating that the mechanical or physical interaction of the SAP155/FIR/FIRΔexon2 complex is potentially essential for sustained expression of both P89 and P27. Together, the interaction between SAP155 and FIR/FIRΔexon2 not only integrates cell-cycle progression and c-Myc transcription by modifying P27 and P89 expression but also suggests that the interaction is a potential target for cancer screening and treatment.

XPB induces C1D expression to counteract UV-induced apoptosis.

Although C1D has been shown to be involved in DNA double-strand break repair, how C1D expression was induced and the mechanism(s) by which C1D facilitates DNA repair in mammalian cells remain poorly understood. We and others have previously shown that expression of xeroderma pigmentosum B (XPB) protein efficiently compensated the UV irradiation-sensitive phenotype of 27-1 cells, which lack functional XPB. To further explore XPB-regulated genes that could be involved in UV-induced DNA repair, differential display analysis of mRNA levels from CHO-9, 27-1, and 27-1 complemented with wild-type XPB was done and C1D gene was identified as one of the major genes whose expression was significantly upregulated by restoring XPB function. We found that XPB is essential to induce C1D transcription after UV irradiation. The increase in C1D expression effectively compensates for the UV-induced proteolysis of C1D and thus maintains cellular C1D level to cope with DNA damage inflicted by UV irradiation. We further showed that although insufficient to rescue 27-1 cells from UV-induced apoptosis by itself, C1D facilitates XPB DNA repair through direct interaction with XPB. Our findings provided direct evidence that C1D is associated with DNA repair complex and may promote repair of UV-induced DNA damage.

[Study on cervical intraepithelial neoplasia in relation to human papillomavirus and co-factors: a case-control study in China and Australia].

OBJECTIVE: This study was to elucidate the role of human papillomavirus (HPV) types and cofactors in the development of cervical intraepithelial neoplasia (CIN). METHODS: Two hundred and twelve women with CIN and 427 women with normal cervical cytology (control group) were recruited from China and Australia. A questionnaire was administered to each participant to obtain the demographic and risk factor information. Cervical biopsies or smears were taken to detect HPV DNA by PCR and to identify HPV types by direct sequencing and/or Amplicor hybridisation. Data were analyzed by logistic regression. RESULTS: HPV prevalence rates of specimens from Chinese and Australian were 11% and 15% among controls (P >0.05), with 99% and 85% of CINs (P<0.001), respectively. The presence of any type of HPV DNA was strongly associated with CIN with OR 43.3 for Chinese and OR 541.6 for Australian women. The strongest risk was for HPV16,followed by HPV31 in Australians, but HPV58, 59 in Chinese women. The risk for multiple HPV infection was stronger in the Australians than that in the Chinese cohort. Except for HPV infection, educational attainment was unexpectedly associated with an increased risk for CIN in Chinese, and cancer history in family was a risk factor for Australians. For the combined cohorts, educational attainment, and frequency of vitamin consumption were identified to be risk factors for CIN. CONCLUSION: Cervical HPV DNA was a major risk factor, with the highest relative risk for type 16 HPV infection for CIN. There were variations in the distribution of HPV genotypes and cofactors in China versus Australia and in CIN.