DamID Research Articles

Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping

How gene positioning to the nuclear periphery regulates transcription remains largely unclear. By cell imaging, we have previously observed the differential compartmentalization of transcription factors and histone modifications at the nuclear periphery in mouse C2C12 myoblasts. Here, we aim to identify DNA sequences associated with the nuclear lamina (NL) and examine this compartmentalization at the genome-wide level. We have integrated high throughput DNA sequencing into the DNA adenine methyltransferase identification (DamID) assay, and have identified ~15, 000 sequencing-based Lamina-Associated Domains (sLADs) in mouse 3T3 fibroblasts and C2C12 myoblasts. These genomic regions range from a few kb to over 1 Mb and cover ~30% of the genome, and are spatially proximal to the NL. Active histone modifications such as H3K4me2/3, H3K9Ac and H3K36me3 are distributed predominantly out of sLADs, consistent with observations from cell imaging that they are localized away from the nuclear periphery. Genomic regions around transcription start sites of expressed sLAD genes display reduced association with the NL; additionally, expressed sLAD genes possess lower levels of active histone modifications than expressed non-sLAD genes. Our work has shown that genomic regions associated with the NL are characterized by the paucity of active histone modifications in mammalian cells, and has revealed novel connections between subnuclear gene positioning, histone modifications and gene expression.

Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping

BackgroundHow gene positioning to the nuclear periphery regulates transcription remains largely unclear. By cell imaging, we have previously observed the differential compartmentalization of transcription factors and histone modifications at the nuclear periphery in mouse C2C12 myoblasts. Here, we aim to identify DNA sequences associated with the nuclear lamina (NL) and examine this compartmentalization at the genome-wide level.ResultsWe have integrated high throughput DNA sequencing into the DNA adenine methyltransferase identification (DamID) assay, and have identified ~15, 000 sequencing-based Lamina-Associated Domains (sLADs) in mouse 3T3 fibroblasts and C2C12 myoblasts. These genomic regions range from a few kb to over 1 Mb and cover ~30% of the genome, and are spatially proximal to the NL. Active histone modifications such as H3K4me2/3, H3K9Ac and H3K36me3 are distributed predominantly out of sLADs, consistent with observations from cell imaging that they are localized away from the nuclear periphery. Genomic regions around transcription start sites of expressed sLAD genes display reduced association with the NL; additionally, expressed sLAD genes possess lower levels of active histone modifications than expressed non-sLAD genes.ConclusionsOur work has shown that genomic regions associated with the NL are characterized by the paucity of active histone modifications in mammalian cells, and has revealed novel connections between subnuclear gene positioning, histone modifications and gene expression.

DNA–protein interactions: methods for detection and analysis

DNA-binding proteins control various cellular processes such as recombination, replication and transcription. This review is aimed to summarize some of the most commonly used techniques to determine DNA-protein interactions. In vitro techniques such as footprinting assays, electrophoretic mobility shift assay, southwestern blotting, yeast one-hybrid assay, phage display and proximity ligation assay have been discussed. The highly versatile in vivo techniques such as chromatin immunoprecipitation and its variants, DNA adenine methyl transferase identification as well as 3C and chip-loop assay have also been summarized. In addition, some in silico tools have been reviewed to provide computational basis for determining DNA-protein interactions. Biophysical techniques like fluorescence resonance energy transfer (FRET) techniques, FRET-FLIM, circular dichroism, atomic force microscopy, nuclear magnetic resonance, surface plasmon resonance, etc. have also been highlighted.

The inner nuclear membrane proteins Man1 and Ima1 link to two different types of chromatin at the nuclear periphery in S. pombe

Metazoan chromatin at the nuclear periphery is generally characterized by lowly expressed genes and repressive chromatin marks and presents a sub-compartment with properties distinct from the nuclear interior. To test whether the S. pombe nuclear periphery behaves similarly, we used DNA adenine methyltransferase identification (DamID) to map the target loci of two inner nuclear membrane proteins, Ima1 and Man1. We found that peripheral chromatin shows low levels of RNA-Polymerase II and nucleosome occupancy, both characteristic of repressed chromatin regions. Consistently, lowly expressed genes preferentially associate with the periphery and highly expressed genes are depleted from it. When looking at peripheral intergenic regions (IGRs), we found that divergent IGRs are enriched compared with convergent IGRs, indicating that transcription preferentially points away from the periphery rather than toward it. Interestingly, we found that Ima1 and Man1 have common, but also separate target regions in the genome. Ima1-interacting loci were enriched for the RNAi components Dcr1 and Rdp1. This agrees with previous findings that Dcr1 is localized at the nuclear periphery. In contrast, Man1 target loci were bound by the heterochromatin protein Swi6, especially at subtelomeric regions. Subtelomeric chromatin was shown to form a unique chromatin type lacking both repressive and active chromatin features and containing low levels of the histone variant H2A.Z. Thus, we find that the fission yeast nuclear periphery shows similar properties to those of metazoan cells, despite the absence of a nuclear lamina. Our results point to a role of nuclear membrane proteins in organizing chromatin domains and loops.

Histone H2B ubiquitylation and H3 lysine 4 methylation prevent ectopic silencing of euchromatic loci important for the cellular response to heat

In Saccharomyces cerevisiae, ubiquitylation of histone H2B signals methylation of histone H3 at lysine residues 4 (K4) and 79. These modifications occur at active genes but are believed to stabilize silent chromatin by limiting movement of silencing proteins away from heterochromatin domains. In the course of studying atypical phenotypes associated with loss of H2B ubiquitylation/H3K4 methylation, we discovered that these modifications are also required for cell wall integrity at high temperatures. We identified the silencing protein Sir4 as a dosage suppressor of loss of H2B ubiquitylation, and we showed that elevated Sir4 expression suppresses cell wall integrity defects by inhibiting the function of the Sir silencing complex. Using comparative transcriptome analysis, we identified a set of euchromatic genes-enriched in those required for the cellular response to heat-whose expression is attenuated by loss of H2B ubiquitylation but restored by disruption of Sir function. Finally, using DNA adenine methyltransferase identification, we found that Sir3 and Sir4 associate with genes that are silenced in the absence of H3K4 methylation. Our data reveal that H2B ubiquitylation/H3K4 methylation play an important role in limiting ectopic association of silencing proteins with euchromatic genes important for cell wall integrity and the response to heat.

Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology

Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.

Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe

In the fission yeast S. pombe, the RNA interference (RNAi) pathway is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic silencing of centromeric repeats. Here, we demonstrate that RNAi also functions to repress genomic elements other than constitutive heterochromatin. Using DNA adenine methyltransferase identification (DamID), we show that the RNAi proteins Dcr1 and Rdp1 physically associate with some euchromatic genes, noncoding RNA genes and retrotransposon long terminal repeats, and that this association is independent of the Clr4 histone methyltransferase. Physical association of RNAi with chromatin is sufficient to trigger a silencing response but not to assemble heterochromatin. The mode of silencing at the newly identified RNAi targets is consistent with a co-transcriptional gene silencing model, as proposed earlier, and functions with trace amounts of siRNAs. We anticipate that similar mechanisms could also be operational in other eukaryotes.

DamID in C. elegans reveals longevity‐associated targets of DAF‐16/FoxO

Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e−11). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16∷DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF ‘parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.

Histone H1 binding is inhibited by histone variant H3.3

Linker histones are involved in the formation of higher-order chromatin structure and the regulation of specific genes, yet it remains unclear what their principal binding determinants are. We generated a genome-wide high-resolution binding map for linker histone H1 in Drosophila cells, using DamID. H1 binds at similar levels across much of the genome, both in classic euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites for active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes; rather, all regions with low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3 causes H1 levels to increase at these sites, with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1, providing a mechanism to keep diverse genomic sites in an open chromatin conformation.

Transcriptional autoregulation in development

The authors would like to thank Peter Harte and Mark Ptashne for helpful and stimulating comments on the manuscript. Our lab's research is supported by grants from the NIH and NSF. The UNC Developmental Biology NIH training grant provided support to J.C.P.

A journey into the nucleus

The EMBO Conference on Nuclear Structure and Dynamics took place between 1 and 5 September 2007, in Montpellier, France, and was organized by G. Cavalli, C. Cremer, S. Gasser, T. Pederson, F. Uhlmann and R. van Driel. ![][1] The basic processes that are responsible for the maintenance and expression of the genome are relatively well understood at the biochemical level, but we still have much to learn about how these processes occur in their physiological context—the cell nucleus—and how they are regulated and coordinated. The second EMBO Conference on Nuclear Structure and Dynamics brought together 240 scientists to discuss the interplay between nuclear structure and genome function. The scope of the meeting was broad, and the speakers covered various topics and experimental approaches. Here, we highlight some of the main issues that were discussed at the conference. Various types of chromatin are segregated in the nucleus. Heterochromatin—which is gene‐poor, rich in proteins that promote gene silencing and characterized by low levels of transcriptional activity—is typically located near the nuclear envelope and around the nucleolus. For this reason, the nuclear periphery has been traditionally regarded as a repressive compartment for gene expression. However, recent studies have shown that the association of actively transcribed genes with nuclear pores gives rise to high levels of expression (for example, Taddei et al , 2006). By using the DNA adenine methyltransferase identification (DamID) technique, B. Van Steensel (Amsterdam, The Netherlands) identified approximately 1,300 lamin‐associated domains (LADs) in human cells and showed that such chromatin regions are characterized by low gene density. LAD borders are flanked by promoters or by CCCTC‐binding factor (CTCF)‐binding sites that potentially act as insulators. B. Kalverda (Amsterdam, The Netherlands) analysed sequences associated with nuclear‐pore proteins in Drosophila cells. In contrast to the lamin‐interacting DNA, the nucleoporin‐interacting sequences contain highly expressed genes, and are … [1]: /embed/graphic-1.gif

A Myc–Groucho complex integrates EGF and Notch signaling to regulate neural development

Integration of patterning cues via transcriptional networks to coordinate gene expression is critical during morphogenesis and misregulated in cancer. Using DNA adenine methyltransferase (Dam)ID chromatin profiling, we identified a protein-protein interaction between the Drosophila Myc oncogene and the Groucho corepressor that regulates a subset of direct dMyc targets. Most of these shared targets affect fate or mitosis particularly during neurogenesis, suggesting the dMyc-Groucho complex may coordinate fate acquisition with mitotic capacity during development. We find an antagonistic relationship between dMyc and Groucho that mimics the antagonistic interactions found for EGF and Notch signaling: dMyc is required to specify neuronal fate and enhance neuroblast mitosis, whereas Groucho is required to maintain epithelial fate and inhibit mitosis. Our results suggest that the dMyc-Groucho complex defines a previously undescribed mechanism of Myc function and may serve as the transcriptional unit that integrates EGF and Notch inputs to regulate early neuronal development.

Chromatin profiling in model organisms

The correct control of gene expression is essential for the proper development of organisms. Abnormal expression of genes can lead to cancerous growth and certain diseases. To understand how gene expression is controlled on a genome-wide scale, methods for assaying transcription factor binding sites are required. There are two prevailing techniques for mapping protein-chromatin interactions, ChIP (chromatin immunoprecipitation) and DamID (DNA adenine methyltransferase identification). Both of these methods, when combined with microarray technology, can provide powerful insights into transcription factor function, higher order chromatin structure and gene regulatory networks. In vivo chromatin profiling studies are now being performed on model organisms, targeting specific tissues to help generate more accurate maps of protein-DNA interactions.

Poor inheritance of low attractiveness for Amblyomma variegatum in cattle

Because of the high variability of cattle infestation, selective breeding has been proposed to control three-host ticks in Africa in the same way as it has been implemented to control the one-host tick Boophilus microplus in Australia. Interbreeding (divergent selection experiment) between Gudali zebu cattle was performed at the Wakwa Research Centre, Adamawa, Cameroon to assess the feasibility and impact of such a selective breeding programme. Fifty cows (chosen out of 129) and 2 bulls (chosen out of 10) were selected according to their greater or lesser attractiveness for the tick Amblyomma variegatum, assessed by their infestation degree (ID, ratio between individual animal infestation and mean herd infestation). Half of the animals displayed a high ID (H) and the remainder a low ID (L). The presence of a suckling calf had a significant effect on the infestation of its dam, the udder of lactating cows being significantly less infested ( P < 0.001) because of a tick removal effect by the calves. Observed infestation data were therefore corrected to allow comparison between lactating and non-lactating cows. The ID of the cows was assessed on five occasions from 1992 to 1994: the consistency of the infestation hierarchy was good overall ( P < 0.001) despite high variability seen in some animals which would complicate the selection of cattle with low infestation. Four breeding groups were set up (L × L, L × H, H × L, H × H) and 40 calves were born in 1993 and 1994. There was a correlation ( P < 0.02–0.001) between liveweight (or age) and ID when the younger calves of the groups were 6 months old, but the correlation disappeared by the time the calves were all older than 1 year. There was no difference between the average ID of the calves from the four breeding groups, and no correlation was observed between dam ID and calf ID. The average ID of the 10 calves born of the dams with the lowest ID was however significantly lower than that of the 10 calves born of the dams with the highest ID (0.82 versus 1.15, P = 0.03), but the difference was much less than that observed between the dams (0.35 versus 2.30, P = 7 × 10 −9). A positive correlation between dam ID and calf ID was found when only these 20 cows were taken into consideration ( r = 0.57, P < 0.01). These results indicate that selective breeding of Gudali zebu cattle to produce animals with low infestation by the tick A. variegatum would be uncertain but that a more drastic selection of the breeders than that implemented in the present study may perhaps have some positive impact.

High-Resolution Mapping Reveals Links of HP1 with Active and Inactive Chromatin Components

Heterochromatin protein 1 (HP1) is commonly seen as a key factor of repressive heterochromatin, even though a few genes are known to require HP1-chromatin for their expression. To obtain insight into the targeting of HP1 and its interplay with other chromatin components, we have mapped HP1-binding sites on Chromosomes 2 and 4 in Drosophila Kc cells using high-density oligonucleotide arrays and the DNA adenine methyltransferase identification (DamID) technique. The resulting high-resolution maps show that HP1 forms large domains in pericentric regions, but is targeted to single genes on chromosome arms. Intriguingly, HP1 shows a striking preference for exon-dense genes on chromosome arms. Furthermore, HP1 binds along entire transcription units, except for 5′ regions. Comparison with expression data shows that most of these genes are actively transcribed. HP1 target genes are also marked by the histone variant H3.3 and dimethylated histone 3 lysine 4 (H3K4me2), which are both typical of active chromatin. Interestingly, H3.3 deposition, which is usually observed along entire transcription units, is limited to the 5′ ends of HP1-bound genes. Thus, H3.3 and HP1 are mutually exclusive marks on active chromatin. Additionally, we observed that HP1-chromatin and Polycomb-chromatin are nonoverlapping, but often closely juxtaposed, suggesting an interplay between both types of chromatin. These results demonstrate that HP1-chromatin is transcriptionally active and has extensive links with several other chromatin components.

The homeobox transcription factor Even-skipped regulates acquisition of electrical properties in Drosophila neurons

BackgroundWhile developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Early decisions, including motoneuron axon targeting, are orchestrated by a cohort of transcription factors that act together in a combinatorial manner. These transcription factors include Even-skipped (Eve), islet and Lim3. The perdurance of these factors in late embryonic neurons is, however, indicative that they might also regulate additional aspects of neuron development, including the acquisition of electrical properties.ResultsTo test the hypothesis that a combinatorial code transcription factor is also able to influence the acquisition of electrical properties in embryonic neurons we utilized the molecular genetics of Drosophila to manipulate the expression of Eve in identified motoneurons. We show that increasing expression of this transcription factor, in two Eve-positive motoneurons (aCC and RP2), is indeed sufficient to affect the electrical properties of these neurons in early first instar larvae. Specifically, we observed a decrease in both the fast K+ conductance (IKfast) and amplitude of quantal cholinergic synaptic input. We used charybdotoxin to pharmacologically separate the individual components of IKfast to show that increased Eve specifically down regulates the Slowpoke (a BK Ca2+-gated potassium channel), but not Shal, component of this current. Identification of target genes for Eve, using DNA adenine methyltransferase identification, revealed strong binding sites in slowpoke and nAcRα-96Aa (a nicotinic acetylcholine receptor subunit). Verification using real-time PCR shows that pan-neuronal expression of eve is sufficient to repress transcripts for both slo and nAcRα-96Aa.ConclusionTaken together, our findings demonstrate, for the first time, that Eve is sufficient to regulate both voltage- and ligand-gated currents in motoneurons, extending its known repertoire of action beyond its already characterized role in axon guidance. Our data are also consistent with a common developmental program that utilizes a defined set of transcription factors to determine both morphological and functional neuronal properties.

DamID, a new tool for studying plant chromatin profiling in vivo, and its use to identify putative LHP1 target loci

We show here that the in vivo methylation-based tagging technique DamID (DNA adenine methyltransferase identification) can be used for studies of DNA-protein interactions or chromatin profiling in plants. We have demonstrated the feasibility, reproducibility and sensitivity of the method in Arabidopsis thaliana, using the well-known yeast GAL4 transcription factor, for which DNA-binding sites (UAS(G)) were introduced into the plant genome. We monitored the methylation resulting from the activity of DNA adenine methyltransferase fused to the protein of interest, by combining digestion with methylation-sensitive restriction enzymes and quantitative PCR. We then used DamID to identify genomic targets of LHP1, a protein mostly associated with euchromatin. We showed that LHP1 was targeted to the promoter and transcribed regions of four genes: AG, AP3, FT and PI. Our data also demonstrate that LHP1, like its animal homologues, has a high binding affinity for A/T-rich regions, binding particularly strongly to the large regulatory introns of AG and PI. We identified three major characteristics of LHP1 binding, highlighting the similarities between plant LHP1 and animal HP1 proteins.

Epigenetics: The Science of Change

For nearly a century after the term “epigenetics” first surfaced on the printed page, researchers, physicians, and others poked around in the dark crevices of the gene, trying to untangle the clues that suggested gene function could be altered by more than just changes in sequence. Today, a wide variety of illnesses, behaviors, and other health indicators already have some level of evidence linking them with epigenetic mechanisms, including cancers of almost all types, cognitive dysfunction, and respiratory, cardiovascular, reproductive, autoimmune, and neurobehavioral illnesses. Known or suspected drivers behind epigenetic processes include many agents, including heavy metals, pesticides, diesel exhaust, tobacco smoke, polycyclic aromatic hydrocarbons, hormones, radioactivity, viruses, bacteria, and basic nutrients. In the past five years, and especially in the past year or two, several groundbreaking studies have focused fresh attention on epigenetics. Interest has been enhanced as it has become clear that understanding epigenetics and epigenomics—the genomewide distribution of epigenetic changes—will be essential in work related to many other topics requiring a thorough understanding of all aspects of genetics, such as stem cells, cloning, aging, synthetic biology, species conservation, evolution, and agriculture.

Evaluation of Specific Gravity as a Screening Test for Colostrum

The objective of this study was to determine the usefulness of specific gravity (colostrometer) in the field evaluation of colostral quality. Colostrum samples (n=150) were collected during the summer of 1997 from 4 dairies in Fresno County, California. Information gathered on each sample at harvest included dam ID, lactation number, yield in kg, hours since calving, colostrometer reading, color, and subjective assessment of thickness. Samples were frozen for further analysis. A 250 ml sample was thawed and hydrometer readings taken to the nearest 0.0005 units at three different temperatures (0-1 °C in an ice bath, 20 °C in a constant temperature bath, and 37 °C ina constant temperature bath). Each sample was analyzed for IgG using VMRD, Inc SRID Kits.

LANDSAT-TM identification of the habitats of the cattle tick, Amblyomma variegatum, in Guadeloupe, French Windward Islands

Because of the high variability of cattle infestation, selective breeding has been proposed to control three-host ticks in Africa in the same way as it has been implemented to control the one-host tick Boophilus microplus in Australia. Interbreeding (divergent selection experiment) between Gudali zebu cattle was performed at the Wakwa Research Centre, Adamawa, Cameroon to assess the feasibility and impact of such a selective breeding programme. Fifty cows (chosen out of 129) and 2 bulls (chosen out of 10) were selected according to their greater or lesser attractiveness for the tick Amblyomma variegatum, assessed by their infestation degree (ID, ratio between individual animal infestation and mean herd infestation). Half of the animals displayed a high ID (H) and the remainder a low ID (L). The presence of a suckling calf had a significant effect on the infestation of its dam, the udder of lactating cows being significantly less infested (P < 0.001) because of a tick removal effect by the calves. Observed infestation data were therefore corrected to allow comparison between lactating and non-lactating cows. The ID of the cows was assessed on five occasions from 1992 to 1994: the consistency of the infestation hierarchy was good overall (P < 0.001) despite high variability seen in some animals which would complicate the selection of cattle with low infestation. Four breeding groups were set up (L × L, L × H, H × L, H × H) and 40 calves were born in 1993 and 1994. There was a correlation (P < 0.02–0.001) between liveweight (or age) and ID when the younger calves of the groups were 6 months old, but the correlation disappeared by the time the calves were all older than 1 year. There was no difference between the average ID of the calves from the four breeding groups, and no correlation was observed between dam ID and calf ID. The average ID of the 10 calves born of the dams with the lowest ID was however significantly lower than that of the 10 calves born of the dams with the highest ID (0.82 versus 1.15, P = 0.03), but the difference was much less than that observed between the dams (0.35 versus 2.30, P = 7 × 10−9). A positive correlation between dam ID and calf ID was found when only these 20 cows were taken into consideration (r = 0.57, P < 0.01). These results indicate that selective breeding of Gudali zebu cattle to produce animals with low infestation by the tick A. variegatum would be uncertain but that a more drastic selection of the breeders than that implemented in the present study may perhaps have some positive impact.