Origins and formation of histone methylation across the human cell cycle

The connections between various nuclear processes and specific histone posttranslational modifications are dependent to a large extent on the acquisition of those modifications after histone synthesis. The reestablishment of histone posttranslational modifications after S phase is especially critical for H3K9 and H3K27 trimethylation, both of which are linked with epigenetic memory and must be stably transmitted from one cellular generation to the next. This report uses a proteomic strategy to interrogate how and when the cell coordinates the formation of histone posttranslational modifications during division. Paramount among the findings is that H3K9 and H3K27 trimethylation begins during S phase but is completed only during the subsequent G(1) phase via two distinct pathways from the unmodified and preexisting dimethylated states. In short, we have systematically characterized the temporal origins and methylation pathways for histone posttranslational modifications during the cell cycle.     

Environmental factors and hygiene behaviors associated with facial cleanliness and trachoma in Kongwa,Tanzania

BackgroundHaving a clean face is protective against trachoma. In the past, long distances to water were associated with unclean faces and increased trachoma. Other environmental factors have not been extensively explored. We need improved clarity on the environmental factors associated with facial cleanliness and trachoma prevalence, especially when the disease burden is low.Methodology/Principle findingsA cross-sectional survey focusing on household environments was conducted in all 92 villages in Kongwa, Tanzania, in a random selection of 1798 households. Children aged 0–5 years in these households were examined for facial cleanliness. In each of the 50 randomly-selected villages, 50 children aged 1–9 years were randomly selected and examined for trachoma. In a multivariate model adjusting for child age, we found that children were more likely to have clean faces if the house had a clean yard (OR 1.62, 95% CI 1.37–1.91), an improved latrine (OR 1.11, 95% CI 1.01–1.22), and greater water storage capacity (OR 1.02, 95% CI 1.00–1.04), and if there were clothes washed and drying around the house (OR 1.30, 95% CI 1.09–1.54). However, measures of crowding, wealth, time spent on obtaining water, or the availability of piped water was not associated with clean faces. Using a cleanliness index (clean yard, improved latrine, washing clothes, ≥1 child in the household having a clean face), the community prevalence of trachoma decreased with an increase in the average value of the index (OR 2.28, 95% CI 1.17–4.80).Conclusions/SignificanceAccess to water is no longer a significant limiting factor in children’s facial cleanliness in Kongwa. Instead, water storage capacity and the way that water is utilized are more important in facial cleanliness. A household cleanliness index with a holistic measure of household environment is associated with reduced community prevalence of trachoma.     

Macrophage-specific expression of class A scavenger receptors enhances granuloma formation in the absence of increased lipid deposition.

Class A scavenger receptors (SR-A) have several proposed functions that could impact atherosclerosis and inflammatory processes. To define the function of SR-A in vivo, we created C57BL/6 transgenic mice that expressed bovine SR-A under the control of the restricted macrophage promoter, lysozyme (lyso-bSR-A). bSR-A mRNA was present in cultured peritoneal macrophages of transgenic mice and tissues that contain significant macrophages including spleen, lung, and ileum. Functional overexpression of SR-A was demonstrated in peritoneal macrophages both by augmented cholesterol ester deposition in response to AcLDL and enhanced adhesion in transgenic mice compared with nontransgenic littermates. To determine whether macrophage-specific expression of bSR-A regulated inflammatory responses, granulomas were generated by subcutaneous injection of carrageenan. Granuloma size was significantly increased in lyso-bSR-A transgenic mice compared with wild-type littermates [421 +/- 51 mg (n = 11) vs. 127 +/- 22 mg (n = 10), P < 0.001]. However, the larger granulomas in lyso-bSR-A transgenic mice were only associated with an increase in unesterified cholesterol, and not cholesterol esters. Furthermore, granulomas from transgenic mice had an increase in the number of macrophages within the tissue.Therefore, macrophage expression of bSR-A increased presence of this cell type in granulomas without enhancing the deposition of cholesterol esters, consistent with a role of the adhesive property of the protein.     

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.     

Ionic interactions in the nitrogenase complex. Properties of Fe-protein containing substitutions for Arg-100.

A series of Azotobacter vinelandii strains have been constructed in which the nitrogenase Fe-protein (Av2) was altered by substitutions for Arg-100. This invariant residue is a likely partner in a salt bridge with the MoFe-protein and, in some species, is the site of reversible regulation by ADP-ribosylation (Pope, M. R., Murrell, S. A., and Ludden, P. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3173-3177). Although we find that arginine is the optimum amino acid, other residues in this position could support diazotrophic growth. These results were surprising because Klebsiella pneumoniae Fe-protein substituted by His-100 had been reported to be inactive (Lowery, R. G., Chang, C. L., Davis, L. C., McKenna, M.-C., Stevens, P. J., and Ludden, P. W. (1989) Biochemistry 28, 1206-1212). Two altered Fe-proteins (Av2-R100Y, the tyrosyl form, and Av2-R100H, the histidyl form) were isolated and, in contrast to this earlier report, we found that both had some activity in acetylene reduction. However, both altered proteins exhibited a decreased maximum velocity (35 and 3% of wild type, respectively) and were strongly inhibited by excess MoFe-protein. These adverse activity parameters were also manifest in the increased sensitivity of the altered proteins to inhibition by salts. Indeed, the salt sensitivity of Av2-R100H is so significant that its activity is masked in the normal assay and is easily missed. In addition, for Av2-R100H, substrate reduction is substantially uncoupled from MgATP hydrolysis. These results suggest that substitutions for Arg-100 may decrease the affinity of the Fe-protein for the MoFe-protein prior to electron transfer but increase affinity after electron transfer. Hence, the role of Arg-100 may be to provide the optimum balance in stabilities of these two complexes for maximum efficiency in substrate reduction.     

Evaluation of photography using head-mounted display technology (ICAPS) for district Trachoma surveys

BackgroundAs the prevalence of trachoma declines worldwide, it is becoming increasingly expensive and challenging to standardize graders in the field for surveys to document elimination. Photography of the tarsal conjunctiva and remote interpretation may help alleviate these challenges. The purpose of this study was to develop, and field test an Image Capture and Processing System (ICAPS) to acquire hands-free images of the tarsal conjunctiva for upload to a virtual reading center for remote grading.Methodology/Principal findingsThis observational study was conducted during a district-level prevalence survey for trachomatous inflammation—follicular (TF) in Chamwino, Tanzania. The ICAPS was developed using a Samsung Galaxy S8 smartphone, a Samsung Gear VR headset, a foot pedal trigger and customized software allowing for hands-free photography. After a one-day training course, three trachoma graders used the ICAPS to collect images from 1305 children ages 1–9 years, which were expert-graded remotely for comparison with field grades. In our experience, the ICAPS was successful at scanning and assigning barcodes to images, focusing on the everted eyelid with adequate examiner hand visualization, and capturing images with sufficient detail to grade TF. The percentage of children with TF by photos and by field grade was 5%. Agreement between grading of the images compared to the field grades at the child level was kappa = 0.53 (95%CI = 0.40–0.66). There were ungradable images for at least one eye in 199 children (9.1%), with more occurring in children ages 1–3 (18.5%) than older children ages 4–9 (4.2%) (χ² = 145.3, p<0.001).Conclusions/SignificanceThe prototype ICAPS device was robust, able to image 1305 children in a district level survey and transmit images from rural Tanzania to an online grading platform. More work is needed to improve the percentage of ungradable images and to better understand the causes of disagreement between field and photo grading.     

Serology,infection, and clinical trachoma as tools in prevalence surveys for re-emergence of trachoma in a formerly hyperendemic district

BackgroundTo eliminate trachoma as a public health problem, countries must achieve a district-level prevalence of trachomatous inflammation—follicular (TF) <5% in children ages 1–9 years. Re-emergence of TF could trigger additional rounds of mass drug/antibiotic administration (MDA), so accurate tools for use in surveys assessing trachoma prevalence are essential.Methodology & principal findingsWe surveyed 2401 children ages 1–9 years from 50 villages in Kongwa, Tanzania, 2 years post-MDA and 1.5 years after an impact survey found TF <5% in the same villages. Our survey included multiple tools: clinical determination of TF, Cepheid testing for Chlamydia trachomatis infection, and testing for anti-pgp3 antibodies via multiplex bead array. Photographs of the upper tarsal conjunctiva were taken in a subset of children to corroborate the field grades.Overall TF prevalence in 1–9 year olds was 7.1% (95% CI: 5.6%-8.9%), which decreased with age (p = <0.0001). TF prevalence by village was heterogeneous, with 19 villages having TF <5% and 16 villages having TF >10%. There was a strong correlation between field and photo grading of TF (kappa = 0.69; 95% CI: 0.60–0.78) and between TF and infection, with 21.5% of TF-positive children also testing positive for infection, as compared to only 1.6% of TF-negative children (p = 0.0010). Overall seroprevalence was 18.2% (95% CI: 14.8%-22.1%), which increased with age (p = <0.0001). Notably, 1–2 year olds, who were born after the cessation of MDA and theoretically should not have had exposure to C. trachomatis in the absence of transmission, had an average seroprevalence of 6.7%.Conclusions & significanceField TF prevalence, supported by photographic review and infection data, suggested re-emergence of trachoma in Kongwa. Moreover, seropositivity in the children born after cessation of MDA indicated exposure to C. trachomatis despite a previous survey finding of TF <5%. Examining seropositivity in specific age groups expected to have limited exposure to C. trachomatis can be used to detect re-emergence.     

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.     

Risk factors for the progression of trachomatous scarring in a cohort of women in a trachoma low endemic district in Tanzania

BackgroundTrachoma, a chronic conjunctivitis caused by Chlamydia trachomatis, is the leading infectious cause of blindness worldwide. Trachoma has been targeted for elimination as a public health problem which includes reducing trachomatous inflammation—follicular prevalence in children and reducing trachomatous trichiasis prevalence in adults. The rate of development of trachomatous trichiasis, the potentially blinding late-stage trachoma sequelae, depends on the rate of trachomatous scarring development and progression. Few studies to date have evaluated the progression of trachomatous scarring in communities that have recently transitioned to a low trachomatous inflammation—follicular prevalence.Methodology/Principal findingsWomen aged 15 and older were randomly selected from households in 48 communities within Kongwa district, Tanzania and followed over 3.5 years for this longitudinal study. Trachomatous inflammation—follicular prevalence was 5% at baseline and at follow-up in children aged 1–9 in Kongwa, Tanzania. 1018 women aged 15 and older had trachomatous scarring at baseline and were at risk for trachomatous scarring progression; 691 (68%) completed follow-up assessments. Photographs of the upper tarsal conjunctiva were obtained at baseline and follow-up and graded for trachomatous scarring using a previously published four-step severity scale. The overall cumulative 3.5-year progression rate of scarring was 35.3% (95% CI 31.6–39.1). The odds of TS progression increased with an increase in age in women younger than 50, (OR 1.03, 95% CI 1.01–1.05, p = 0.005) as well as an increase in the household poverty index (OR 1.29, 95% CI 1.13–1.48, p = 0.0002).Conclusions/SignificanceThe 3.5-year progression of scarring among women in Kongwa, a formerly hyperendemic now turned hypoendemic district in central Tanzania, was high despite a low active trachoma prevalence. This suggests that the drivers of scarring progression are likely not related to on-going trachoma transmission in this district.